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Site-Specific Glycan-Peptide Analysis for Determination of N-Glycoproteome Heterogeneity
Abstract
A combined glycomics and glycoproteomics strategy was developed for the site-specific analysis of N-linked glycosylation heterogeneity from a complex mammalian protein mixture. Initially, global characterization of the N-glycome was performed using porous graphitized carbon liquid chromatography - tandem mass spectrometry (PGC-LC-MS/MS) and the data used to create an N-glycan modification database. In the next step, tryptic glycopeptides were enriched using zwitterionic hydrophilic interaction liquid chromatography (Zic-HILIC) and fractionated by reversed-phase liquid chromatography (RPLC; pH 7.9). The resulting fractions were each separated into two equal aliquots. The first set of aliquots were treated with peptide-N-glycosidase F (PNGase F) to remove N-glycans, and the former N-glycopeptides analyzed by nano-RPLC-MS/MS (pH 2.7) and identified by Mascot database search. This enabled the creation of a glycopeptide-centric concatenated database for each fraction. The second set of aliquots was analyzed directly by nanoRPLC-MS/MS (pH 2.7), employing fragmentation by CID and HCD. The assignment of glycan compositions to peptide sequences was achieved by searching the N-glycopeptide HCD MS/MS spectra against the glycopeptide-centric concatenated databases employing the N-glycan modification database. CID spectra were used to assign glycan structures identified in the glycomic analysis to peptide sequences. This multi-dimensional approach allowed confident identification of 863 unique intact N-linked glycopeptides from 161 rat brain glycoproteins.
... Expression of influenza A virus glycan receptor candidates in mallard 3 (HILIC) according to (Parker et al. 2013), with slight modifications. In short, peptides were loaded onto an in-house zwitterionic Zic-HILIC SPE cartridge containing 20 mg of Zic-HILIC particles (10 μm, 200 Å; Sequant/Merck). ...
... We could also characterize 121 O-glycopeptides from 87 glycoproteins with complete information on the Neu5Acα2-3-/ Neu5Acα2-6-linkage status. The HILIC enrichment strategy was developed to enrich for N-glycopeptides (Parker et al. 2013). However, by manual fragmentation analysis of the LC-MS/MS data we also identified fragmentation patterns consistent with mono-and disialo core 1 O-glycopeptides and consequently we also performed an O-glycoproteomic Byonic analysis (Fig. 2 and Supplementary Tables S2-S4). ...
Influenza A virus (IAV) pandemics result from interspecies transmission events within the avian reservoir and further into mammals including humans. Receptor incompatibility due to differently expressed glycan structures between species has been suggested to limit zoonotic IAV transmission from the wild bird reservoir as well as between different bird species. Using glycoproteomics, we have studied the repertoires of expressed glycan structures with focus on putative sialic acid-containing glycan receptors for IAV in mallard, chicken and tufted duck; three bird species with different roles in the zoonotic ecology of IAV. The methodology used pinpoints specific glycan structures to specific glycosylation sites of identified glycoproteins and was also used to successfully discriminate α2-3- from α2-6-linked terminal sialic acids by careful analysis of oxonium ions released from glycopeptides in tandem MS/MS (MS2), and MS/MS/MS (MS3). Our analysis clearly demonstrated that all three bird species can produce complex N-glycans including α2-3-linked sialyl Lewis structures, as well as both N- and O- glycans terminated with both α2-3- and α2-6-linked Neu5Ac. We also found the recently identified putative IAV receptor structures, Man-6P N-glycopeptides, in all tissues of the three bird species. Furthermore, we found many similarities in the repertoires of expressed receptors both between the bird species investigated and to previously published data from pigs and humans. Our findings of sialylated glycan structures, previously anticipated to be mammalian specific, in all three bird species may have major implications for our understanding of the role of receptor incompatibility in interspecies transmission of IAV.
... In recent times, niier bioanalytical methods strongly contributed to better resolve the brain N-glycosylation puzzle, producing reliable data from milligram amounts of rodent brain tissues, greatly reducing the sample preparation time, and oen enhancing N-glycan recovery through PNGase F enzymatic release. [11][12][13][14][15][16][17][18][19] However, some of these bioanalytical methods still present limitations. For instance, some employ hydrazinolysis for N-glycan release, complicating their purication aer a chemical reaction with multiple chromatographic steps, yielding only a few N-glycan structures, 11,13 and degrading the proteins. ...
... This approach led to the identication of 40 to $80 N-glycan monosaccharide compositions from human and mouse brains, respectively, but it employed trypsinisation before enzymatic deglycosylation, therefore adding an overnight step and prolonging the work-ow. Other bioanalytical methods, focusing on the analysis of membrane-associated brain glycoproteins, resolved $70 Nglycan monosaccharide compositions, 12 albeit with a suboptimal mass accuracy (mostly >20 ppm, up to 80 Da delta mass); set the lowest starting amount of brain tissue producing reliable data across the literature (1.5 mg) along with a resolution of up to 120 N-glycan monosaccharide compositions; 15 and efficiently distinguished the relative amounts of majorly expressed isomeric glycotopes with high sensitivity through MS 2 /MS 3 data acquisition. 17 A recently published method developed in our laboratory (from now on called Homogenate in-Solution Deglycosylation, HSD) 18 was the rst one to use Hydrophilic Interaction Liquid Chromatography (HILIC-UPLC) to separate the complex ensemble of N-glycan structures puried from brain tissue, with good sensitivity and resolution, but one limitation of this method is that it is not possible to analyse both N-glycans and proteins from the same sample, thus losing valuable information about the N-glycoprotein component. ...
Glycans influence a variety of aspects of tissue development and pathophysiology, harboring a wealth of biochemical information exploitable for the discovery of new biomarkers. The development of versatile and precise glycoanalytical platforms is crucial to bring to the scientific community reliable tools to unveil the glycan-encoded biochemical information. In this work, a novel platform for brain glycan analysis, named Lysate in-Solution Deglycosylation (LSD), is presented, validated and compared to similar methodologies reported in the literature. Briefly, within the LSD workflow, brain tissue is lysed, proteins are purified via methanol/chloroform extraction, and directly deglycosylated with PNGase F. Released N-glycans are purified using a second round of methanol/chloroform extraction, labelled with fluorescent tags, and cleaned-up prior to LC-FLR/MS analysis. LSD is a quick, sensitive, robust, precise, specific and versatile glycomic workflow, compatible with multiple glyco(proteo)mics analyses from the same sample and eventually applicable to other solid tissues. LSD was devised as a plug-and-play platform for quick glycosylation analysis, and was designed to be compatible with a variety of analytical set-ups (i.e., diverse detection systems), thus enabling the extraction of an additional layer of information from different studies (i.e., proteomics) without the need to collect fresh biological material. In comparison to analogous N-glycomic workflows, LSD performs reliably on the smallest amount of starting brain tissue and, to the best of the authors knowledge, is currently the most thoroughly validated neuro-N-glycomic method in the field-related literature, thus representing a significant step forward on the road towards glycan-based biomedical applications.
... The tryptic digests of 25 μg of protein from the mouse liver were mixed with 2.5 mg of PDA-ADE@KCP in an optimized loading buffer (90% ACN, 1% TFA) and 7.5 mg of ZIC-HILIC in a previously reported optimal loading buffer (80% ACN, 1% TFA) 34 and the mixtures were incubated for 2 hours at 4°C. Next, the mixture was loaded into a tip filled with a C8 membrane at the bottom. ...
An efficient strategy was firstly proposed and successfully applied to simultaneous enrichment of N - and O -glycopeptides from complex biological samples through the synergistic effect of hydrophilic and electrostatic interactions.
... In this study, we undertook a detailed analysis of human GPIbα glycosylation. We purified the GPIbα ectodomain from human platelets and then used a combination of experimental strategies, including mucinase digestion methods, to carry out a glycomics-informed MS glycopeptide analysis [41,42]. By integrating information from glycomics and glycopeptide analysis, we defined glycan structures in detail and site-localized them to specific amino acids. ...
Background:
Platelet glycoprotein (GP) Ibα is the major ligand-binding subunit of the GPIb-IX-V complex that binds von Willebrand factor. GPIbα is heavily glycosylated, and its glycans have been proposed to play key roles in platelet clearance, von Willebrand factor binding, and as target antigens in immune thrombocytopenia syndromes. Despite its importance in platelet biology, the glycosylation profile of GPIbα is not well characterized.
Objectives:
The aim of this study was to comprehensively analyze GPIbα amino acid sites of glycosylation (glycosites) and glycan structures.
Methods:
GPIbα ectodomain that was recombinantly expressed or that was purified from human platelets was analyzed by Western blot, mass spectrometry glycomics, and mass spectrometry glycopeptide analysis to define glycosites and the structures of the attached glycans.
Results:
We identified a diverse repertoire of N- and O-glycans, including sialoglycans, Tn antigen, T antigen, and ABO(H) blood group antigens. In the analysis of the recombinant protein, we identified 62 unique O-glycosites. In the analysis of the endogenous protein purified from platelets, we identified 48 unique O-glycosites and 1 N-glycosite. The GPIbα mucin domain is densely O-glycosylated. Glycosites are also located within the macroglycopeptide domain and mechanosensory domain.
Conclusions:
This comprehensive analysis of GPIbα glycosylation lays the foundation for further studies to determine the functional and structural roles of GPIbα glycans.
... The cleaved samples were desalted with C18 spin columns and dissolved in 80% acetonitrile/1% TFA in water, and glycopeptides enriched with hydrophilic interaction liquid chromatography (HILIC), with slight modifications [40]. Briefly, the samples were applied to in-house packed cartridges containing 20 mg Zic-HILIC particles (10 µm, 200 Å; Sequant/Merck). ...
The Sda histo-blood group antigen (GalNAcβ1-4(NeuAcα2-3)Galβ-R) is implicated in various infections and constitutes a potential biomarker for colon cancer. Sd(a−) individuals (2–4% of Europeans) may produce anti-Sda, which can lead to incompatible blood transfusions, especially if donors with the high-expressing Sd(a++)/Cad phenotype are involved. We previously reported the association of B4GALNT2 mutations with Sd(a−), which established the SID blood-group system. The present study provides causal proof underpinning this correlation. Sd(a−) HEK293 cells were transfected with different B4GALNT2 constructs and evaluated by immunostaining and glycoproteomics. The predominant SIDnull candidate allele with rs7224888:T>C (p.Cys406Arg) abolished Sda synthesis, while this antigen was detectable as N- or O-glycans on glycoproteins following transfection of wildtype B4GALNT2. Surprisingly, two rare missense variants, rs148441237:A>G and rs61743617:C>T, found in a Sd(a−) compound heterozygote, gave results similar to wildtype. To elucidate on whether Sd(a++)/Cad also depends on B4GALNT2 alterations, this gene was sequenced in five individuals. No Cad-specific changes were identified, but a detailed erythroid Cad glycoprotein profile was obtained, especially for glycophorin-A (GLPA) O-glycosylation, equilibrative nucleoside transporter 1 (S29A1) O-glycosylation, and band 3 anion transport protein (B3AT) N-glycosylation. In conclusion, the p.Cys406Arg β4GalNAc-T2 variant causes Sda-deficiency in humans, while the enigmatic Cad phenotype remains unresolved, albeit further characterized.
... Early methods for N-glycoprofiling of brain tissue were lengthy, laborious, and required large amounts of tissue [11][12][13][14][15]. In recent times, the advent of a variety of simpler, quicker, and more sensitive methods [16][17][18][19][20][21][22][23][24][25][26] has paved the way for rapid acceleration in the field of neuroglycomics and a surge in publications that examine the total brain N-glycome and how it changes under various physiological or pathological conditions. Only now do we have the tools to address these kinds of questions and to explore the dynamic brain N-glycome in all its complexity on a reasonable scale [27]. ...
The attachment of carbohydrates to other macromolecules, such as proteins or lipids, is an important regulatory mechanism termed glycosylation. One subtype of protein glycosylation is asparagine-linked glycosylation (N-glycosylation) which plays a key role in the development and normal functioning of the vertebrate brain. To better understand the role of N-glycans in neurobiology, it’s imperative we analyse not only the functional roles of individual structures, but also the collective impact of large-scale changes in the brain N-glycome. The systematic study of the brain N-glycome is still in its infancy and data are relatively scarce. Nevertheless, the prevailing view has been that the neuroglycome is inherently restricted with limited capacity for variation. The development of improved methods for N-glycomics analysis of brain tissue has facilitated comprehensive characterisation of the complete brain N-glycome under various experimental conditions on a larger scale. Consequently, accumulating data suggest that it’s more dynamic than previously recognised and that, within a general framework, it has a given capacity to change in response to both intrinsic and extrinsic stimuli. Here, we provide an overview of the many factors that can alter the brain N-glycome, including neurodevelopment, ageing, diet, stress, neuroinflammation, injury, and disease. Given this emerging evidence, we propose that the neuroglycome has a hitherto underappreciated plasticity and we discuss the therapeutic implications of this regarding the possible reversal of pathological changes via interventions. We also briefly review the merits and limitations of N-glycomics as an analytical method before reflecting on some of the outstanding questions in the field.
Graphic abstract
... To address this, various chromatographic methods (lectin affinity chromatography [LAC], hydrazide chemistry, boronate affinity chromatography [BAC], and hydrophilic interaction liquid chromatography (HILIC)) have been widely used to selectively capture the glycoproteins or glycopeptides [18][19][20][21][22][23][24][25][26]. Readers are referred to references [25][26][27][28] to understand the principles of the above-mentioned chromatography methods. ...
Among various protein post‐translational modifications (PTMs), glycosylation has received special attention due to its immense role in molecular interactions, cellular signal transduction, immune response etc. Aberration in glycan moieties of a glycoprotein is associated with cancer, diabetes, and bacterial and viral infections. In biofluids (plasma, saliva, urine, milk etc.), glycoproteins are low in abundance and are masked by the presence of high abundant proteins. Hence, prior to their identification using mass spectrometry (MS) methods, liquid chromatography (LC) based approaches were widely used. A general enrichment strategy involves a protein digestion step, followed by LC based enrichment and desorption of glycopeptides, and enzymatic excision of the glycans. The focus of this review paper is to highlight the articles published since 2016 that dealt with different LC based approaches for glycopeptide and glycoprotein enrichment. The preparation of stationary phases, their surface activation, and ligand immobilization strategies have been discussed in detail. Finally, the major developments and future trends in the field have been summarized. This article is protected by copyright. All rights reserved
... For reduction of computing time, an initial global characterization of the Nglycome can be performed by CE. The resulting glycan list is used to search against a targeted set of defined N-glycan structures as variable protein/peptide modifications (Thaysen-Andersen and Packer 2014;Parker et al. 2013;Lebede et al. 2021;Pralow et al. 2021;Pioch et al. 2018) ...
The in-depth characterization of protein glycosylation has become indispensable in many research fields and in the biopharmaceutical industry. Especially knowledge about modulations in immunoglobulin G (IgG) N-glycosylation and their effect on immunity enabled a better understanding of human diseases and the development of new, more effective drugs for their treatment. This chapter provides a deeper insight into capillary (gel) electrophoresis-based (C(G)E) glycan analysis, addressing its impressive performance and possibilities, its great potential regarding real high-throughput for large cohort studies, as well as its challenges and limitations. We focus on the latest developments with respect to miniaturization and mass spectrometry coupling, as well as data analysis and interpretation. The use of exoglycosidase sequencing in combination with current C(G)E technology is discussed, highlighting possible difficulties and pitfalls. The application section describes the detailed characterization of N-glycosylation, utilizing multiplexed CGE with laser-induced fluorescence detection (xCGE-LIF). Besides a comprehensive overview on antibody glycosylation by comparing species-specific IgGs and human immunoglobulins A, D, E, G, and M, the chapter comprises a comparison of therapeutic monoclonal antibodies from different production cell lines, as well as a detailed characterization of Fab and Fc glycosylation. These examples illustrate the full potential of C(G)E, resolving the smallest differences in sugar composition and structure.
... Currently, liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is the method of choice widely used in proteomics and glycoproteomics 11,12 . Novel MS/MS fragmentation methods derived from higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD), such as stepped collision energy HCD (SCE-HCD) 13 and ETD with supplemental HCD (EThcD) 14 , have been proven powerful for intact glycopeptides profiling. ...
Large-scale profiling of intact glycopeptides is critical but challenging in glycoproteomics. Data independent acquisition (DIA) is an emerging technology with deep proteome coverage and accurate quantitative capability in proteomics studies, but is still in the early stage of development in the field of glycoproteomics. We propose GproDIA, a framework for the proteome-wide characterization of intact glycopeptides from DIA data with comprehensive statistical control by a 2-dimentional false discovery rate approach and a glycoform inference algorithm, enabling accurate identification of intact glycopeptides using wide isolation windows. We further utilize a semi-empirical spectrum prediction strategy to expand the coverage of spectral libraries of glycopeptides. We benchmark our method for N-glycopeptide profiling on DIA data of yeast and human serum samples, demonstrating that DIA with GproDIA outperforms the data-dependent acquisition-based methods for glycoproteomics in terms of capacity and data completeness of identification, as well as accuracy and precision of quantification. We expect that this work can provide a powerful tool for glycoproteomic studies. Data independent acquisition (DIA) proteomics provides deep coverage and high quantitative accuracy, but is not yet well established in glycoproteomics. Here, the authors develop a DIA-based glycoproteomics workflow with stringent statistical controls to enable accurate glycopeptide identification.
... Protein glycosylation, a representative type of common and heterogeneous post-translational modifications (PTMs) [1,2], participates in biological events [3,4] and alters the functions of proteins [5]. The analysis of glycan structure and glycosite is very significant to fully and accurately understand the role of them in glycosylation function [6][7][8]. Mass spectrometry (MS) has developed into the most popular instrument for glycopeptide identification, but the analysis of intact glycopeptides using MS is a challenging issue due to the low abundance, complexity of the co-existed matrixes, and heterogeneity [9]. Before mass spectrometry analysis, it is necessary to explore efficient isolation ways that can retrieve glycopeptides intact, which is essential for the identification of glycopeptides [10,11]. ...
A novel type of boric acid–functionalized magnetic covalent organic framework (mCOF) with polyethyleneimine (PEI) as a linker (denoted as mCOF@PEI@B(OH)2) has been prepared through a post-synthesis strategy, which points out an achievable path for the construction of boronic acid–functionalized COFs. Based on the boric acid chemistry, the obtained core-shell structured mCOF@PEI@B(OH)2 can selectively isolate glycopeptides through the modified boronic acid groups. The mCOF@PEI@B(OH)2 exhibits excellent performance with good reusability (ten cycles), low detection limit (0.5 fmol·μL⁻¹), size-exclusion effect, and relatively high loading capacity (80 μg·mg⁻¹), recovery yield (94.9 ± 2.8%), and selectivity (HRP digests:BSA digests = 1:500). Detection is done by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In addition, 37 endogenous glycopeptides are captured from human saliva with mCOF@PEI@B(OH)2, providing effective proofs for its capability to capture low-abundance glycopeptides from actual biological samples.
Graphical abstract
... In the authors' experience, the ability to incorporate glycomics data into the glycoproteomics workflow, coined as "glycomics-assisted glycoproteomics", is an example of a particular useful integration of multiple-omics data sets. These allow an informed definition of the glycan search space whilst providing detailed information on the attached glycan structures [79,126,129,141,[178][179][180]. Notably, careful manual review of the data output is still needed to obtain reliable and reproducible results in large-scale glycopeptide data analysis. ...
Protein glycosylation is one of the most common post-translational modifications that are essential for cell function across all domains of life. Changes in glycosylation are considered a hallmark of many diseases, thus making glycoproteins important diagnostic and prognostic biomarker candidates and therapeutic targets. Glycoproteomics, the study of glycans and their carrier proteins in a system-wide context, is becoming a powerful tool in glycobiology that enables the functional analysis of protein glycosylation. This 'Hitchhiker's guide to glycoproteomics' is intended as a starting point for anyone who wants to explore the emerging world of glycoproteomics. The review moves from the techniques that have been developed for the characterisation of single glycoproteins to technologies that may be used for a successful complex glycoproteome characterisation. Examples of the variety of approaches, methodologies, and technologies currently used in the field are given. This review introduces the common strategies to capture glycoprotein-specific and system-wide glycoproteome data from tissues, body fluids, or cells, and a perspective on how integration into a multi-omics workflow enables a deep identification and characterisation of glycoproteins - a class of biomolecules essential in regulating cell function.
... Therefore, the related functional glycoproteins and detailed mechanism are still largely unknown. In recent years, site-specific glycosylation analysis has been achieved by directly analyzing intact glycopeptides using mass spectrometry [21][22][23][24][25], by which both glycan alterations and their attached glycoprotein information can be clearly profiled. We reason that this glycoproteomic technology has great chance to recover the possible connections between altered glycans and glycosylated proteins associated with EMT of cancer cells, and even identify some new site-specific glycan changes related to EMT to facilitate a better understanding of the EMT mechanism. ...
Rationale: Epithelial-mesenchymal transition (EMT) has been recognized as an important step toward high invasion and metastasis of many cancers including hepatocellular carcinoma (HCC), while the mechanism for EMT promotion is still ambiguous.
Methods: The dynamic alterations of site-specific glycosylation during HGF/TGF-β1-induced EMT process of three HCC cell lines were systematically investigated using precision glycoproteomic methods. The possible roles of EMT-related glycoproteins and site-specific glycans were further confirmed by various molecular biological approaches.
Results: Using mass spectrometry-based glycoproteomic methods, we totally identified 2306 unique intact glycopeptides from SMMC-7721 and HepG2 cell lines, and found that core-fucosylated glycans were accounted for the largest proportion of complex N-glycans. Through quantification analysis of intact glycopeptides, we found that the majority of core-fucosylated intact glycopeptides from folate receptor α (FOLR1) were up-regulated in the three HGF-treated cell lines. Similarly, core-fucosylation of FOLR1 were up-regulated in SMMC-7721 and Hep3B cells with TGF-β1 treatment. Using molecular approaches, we further demonstrated that FUT8 was a driver for HGF/TGF-β1-induced EMT. The silencing of FUT8 reduced core-fucosylation and partially blocked the progress of HGF-induced EMT. Finally, we confirmed that the level of core-fucosylation on FOLR1 especially at the glycosite Asn-201 positively regulated the cellular uptake capacity of folates, and enhanced uptake of folates could promote the EMT of HCC cells.
Conclusions: Based on the results, we proposed a potential pathway for HGF or TGF-β1-induced EMT of HCC cells: HGF or TGF-β1 treatment of HCC cells can increase the expression of glycosyltransferase FUT8 to up-regulate the core-fucosylation of N-glycans on glycoproteins including the FOLR1; core-fucosylation on FOLR1 can then enhance the folate uptake capacity to finally promote the EMT progress of HCC cells.
... HILIC does not discriminate between O-linked and N-linked glycopeptides and hydrophilic non-glycosylated peptides may co-elute [20]. Thus, for more complete glycopeptide enrichment, HILIC may require a complementary chromatography fractionation step [21]. ...
High confidence methods are needed for determining the glycosylation profiles of complex biological samples as well as recombinant therapeutic proteins. A common glycan analysis workflow involves liberation of N-glycans from glycoproteins with PNGase F or O-glycans by hydrazinolysis prior to their analysis. This method is limited in that it does not permit determination of glycan attachment sites. Alternative proteomics-based workflows are emerging that utilize site-specific proteolysis to generate peptide mixtures followed by selective enrichment strategies to isolate glycopeptides. Methods designed for the analysis of complex samples can yield a comprehensive snapshot of individual glycans species, the site of attachment of each individual glycan and the identity of the respective protein in many cases. This chapter will highlight advancements in enzymes that digest glycoproteins into distinct fragments and new strategies to enrich specific glycopeptides.
... Currently, liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is the method of choice widely used in proteomics and glycoproteomics 11,12 . ...
Large-scale profiling of intact glycopeptides is critical but challenging in glycoproteomics. Data independent acquisition (DIA) is an emerging technology with deep proteome coverage and accurate quantitative capability in proteomics studies, but is still in the early stage of development in the field of glycoproteomics. We propose GproDIA, a framework for the proteome-wide characterization of intact glycopeptides from DIA data with comprehensive statistical control by a 2-dimentional false discovery rate approach and a glycoform inference algorithm, enabling accurate identification of intact glycopeptides using wide isolation windows. We further adapt a semi-empirical spectrum prediction strategy to expand the coverage of spectral libraries of glycopeptides. We benchmark our method for N-glycopeptide profiling on DIA data of yeast and human serum samples, demonstrating that DIA with GproDIA outperforms the data dependent acquisition (DDA) based methods for glycoproteomics in terms of capacity and data completeness of identification, as well as accuracy and precision of quantification. We expect that this work can provide a powerful tool for glycoproteomic studies.
... High mannose N-glycans are found in the surface of endothelial cells and participate in some molecular processes of cell interaction and recognition (Gu et al., 2012;Schwarz & Aebi, 2011). MAN3, MAN5 and MAN9 glycans, for example, are anchored to glycoproteins, most of which are involved in cell recognition processes, immune system regulation and other metabolic processes (Parker et al., 2013;Rudd, 2001;Sparrow et al., 2008). Some biological effects, such as inflammation (Pinto-Junior et al., 2017a) and vasorelaxation, may be related to interactions of the lectin with glycosylated receptors in endothelial cells. ...
Lectins are a class of proteins or glycoproteins capable of recognizing and interacting with carbohydrates in a specific and reversible manner. Owing to this property, these proteins can interact with glycoconjugates present on the cell surface, making it possible to decipher the glycocode, as well as elicit biological effects, such as inflammation and vasorelaxation. Here, we report a structural and biological study of the mannose/glucose‐specific lectin from Dioclea lasiophylla seeds, DlyL. The study aimed to evaluate in detail the interaction of DlyL with Xman and high-mannose N-glycans (MAN3, MAN5 and MAN9) by molecular dynamics (MD) and the resultant in vitro effect on vasorelaxation using rat aortic rings. In silico analysis of molecular docking was performed to obtain the initial coordinates of the DlyL complexes with the carbohydrates to apply as inputs in MD simulations. The MD trajectories demonstrated the stability of DlyL over time as well as different profiles of interaction with Xman and N-glycans. Furthermore, aortic rings assays demonstrated that the lectin could relax pre-contracted aortic rings with the participation of the carbohydrate recognition domain (CRD) and nitric oxide (NO) when endothelial tissue is preserved. These results confirm the ability of DlyL to interact with high‐mannose N‐glycans with its expanded CRD, supporting the hypothesis that DlyL vasorelaxant activity occurs primarily through its interaction with cell surface glycosylated receptors.
Communicated by Ramaswamy H. Sarma
... Robust probability-based false discovery rate-driven search engines and algorithms for intact glycopeptides are still required to propel the field forward. Adding to the informatics advances needed to achieve such important improvements, 'glycomics-assisted glycoproteomics' represents a complementary strategy that can guide the glycoproteomics data analysis to lower the glycopeptide false discovery rates while simultaneously addressing the complexity of the glycome [35,43,67,246,247]. ...
Facilitated by advances in the separation sciences, mass spectrometry and informatics, glycoproteomics, the analysis of intact glycopeptides at scale, has recently matured enabling new insights into the complex glycoproteome. While diverse quantitative glyco-proteomics strategies capable of mapping monosaccharide compositions of N-and O-linked glycans to discrete sites of proteins within complex biological mixtures with considerable sensitivity, quantitative accuracy and coverage have become available, developments supporting the advancement of structure-focused glycoproteomics, a recognised frontier in the field, have emerged. Technologies capable of providing site-specific information of the glycan fine structures in a glycoproteome-wide context are indeed necessary to address many pending questions in glycobiology. In this review, we firstly survey the latest glycoproteomics studies published in 2018-2020, their approaches and their findings, and then summarise important technological innovations in structure-focused glycoproteomics. Our review illustrates that while the O-glycoproteome remains comparably under-explored despite the emergence of new O-glycan-selective mucinases and other innovative tools aiding O-glycoproteome profiling, quantitative glycoproteomics is increasingly used to profile the N-glycoproteome to tackle diverse biological questions. Excitingly, new strategies compatible with structure-focused glycoproteomics including novel chemoenzymatic labelling, enrichment, separation, and mass spectrometry-based detection methods are rapidly emerging revealing glycan fine structural details including bisecting GlcNAcylation, core and antenna fucosylation, and sialyl-linkage information with protein site resolution. Glycoproteomics has clearly become a mainstay within the glycosciences that continues to reach a broader community. It transpires that structure-focused glycoproteomics holds a considerable potential to aid our understanding of systems glycobiology and unlock secrets of the glycoproteome in the immediate future.
... Here, we present the systematic proteomic and glycoproteomic analyses of 83 prospectively collected HGSC tissues and 23 relevant non-tumor tissues. The analysis allowed us to identify and quantify sites of N-linked glycosylation using a Solid-Phase Extraction of Glycosite-containing peptides (SPEG) approach (Zhang et al., 2003), as well as identification of the glycans occupying those N-linked sites using intact glycopeptides (IGPs) analysis (Khatri et al., 2016;Parker et al., 2013;Scott et al., 2011;Sun et al., 2016). This integrated approach provides a comprehensive look at the N-glycosylated proteins, their N-glycosylated sites, and the glycans occupying these sites in one study ( Figure 1A). ...
Many gene products exhibit great structural heterogeneity because of an array of modifications. These modifications are not directly encoded in the genomic template but often affect the functionality of proteins. Protein glycosylation plays a vital role in proper protein functions. However, the analysis of glycoproteins has been challenging compared with other protein modifications, such as phosphorylation. Here, we perform an integrated proteomic and glycoproteomic analysis of 83 prospectively collected high-grade serous ovarian carcinoma (HGSC) and 23 non-tumor tissues. Integration of the expression data from global proteomics and glycoproteomics reveals tumor-specific glycosylation, uncovers different glycosylation associated with three tumor clusters, and identifies glycosylation enzymes that were correlated with the altered glycosylation. In addition to providing a valuable resource, these results provide insights into the potential roles of glycosylation in the pathogenesis of HGSC, with the possibility of distinguishing pathological outcomes of ovarian tumors from non-tumors, as well as classifying tumor clusters.
... HILIC enrichments have been used to enrich glycopeptides from a wide range of biological matrices, including human biofluids (e.g., plasma, serum, and milk) (209)(210)(211)(212)(213)(214), cancer systems (215)(216)(217)(218)(219)(220)(221), other human model systems (222, 223), murine tissues (178, [224][225][226][227], pathogens (228)(229)(230)(231), and plants (232). HILIC has been used both standalone and in combination with IMAC and MOAC to enrich phospho-and glycopeptides, too, as mentioned above. ...
Glycosylation is a prevalent, yet heterogeneous modification with a broad range of implications in molecular biology. This heterogeneity precludes enrichment strategies that can be universally beneficial for all glycan classes. Thus, choice of enrichment strategy has profound implications on experimental outcomes. Here we review common enrichment strategies used in modern mass spectrometry (MS)-based glycoproteomic experiments, including lectins and other affinity chromatographies, hydrophilic interaction chromatography (HILIC) and its derivatives, porous graphitic carbon (PGC), reversible and irreversible chemical coupling strategies, and chemical biology tools that often leverage bioorthogonal handles. Interest in glycoproteomics continues to surge as MS instrumentation and software improve, so this review aims to help equip researchers with necessary information to choose appropriate enrichment strategies that best complement these efforts.
... Only HCD-MS/MS spectra containing HexNAc oxonium ions (138.05-138.06 and 204.08-204.09 m/z) were annotated in the final list of glycopeptides as previously described (29). ...
Many cell surface and secreted proteins are modified by the covalent addition of glycans that play an important role in the development of multicellular organisms. These glycan modifications enable communication between cells and the extracellular matrix via interactions with specific glycan-binding lectins and the regulation of receptor-mediated signaling. Aberrant protein glycosylation has been associated with the development of several muscular diseases suggesting essential glycan- and lectin-mediated functions in myogenesis and muscle development but our molecular understanding of the precise glycans, catalytic enzymes and lectins involved remain only partially understood. Here, we quantified dynamic remodeling of the membrane-associated proteome during a time-course of myogenesis in cell culture. We observed wide-spread changes in the abundance of several important lectins and enzymes facilitating glycan biosynthesis. Glycomics-based quantification of released N-linked glycans confirmed remodeling of the glycome consistent with the regulation of glycosyltransferases and glycosidases responsible for their formation including a previously unknown di-galactose-to-sialic acid switch supporting a functional role of these glycoepitopes in myogenesis. Furthermore, dynamic quantitative glycoproteomic analysis with multiplexed stable isotope labelling and analysis of enriched glycopeptides with multiple fragmentation approaches identified glycoproteins modified by these regulated glycans including several integrins and growth factor receptors. Myogenesis was also associated with the regulation of several lectins most notably the up-regulation of galectin-1 (LGALS1). CRISPR/Cas9-mediated deletion of Lgals1 inhibited differentiation and myotube formation suggesting an early functional role of galectin-1 in the myogenic program. Importantly, similar changes in N-glycosylation and the up-regulation of galectin-1 during postnatal skeletal muscle development were observed in mice. Treatment of new-born mice with recombinant adeno-associated viruses to overexpress galectin-1 in the musculature resulted in enhanced muscle mass. Our data form a valuable resource to further understand the glycobiology of myogenesis and will aid the development of intervention strategies to promote healthy muscle development or regeneration.
... Site-specific glycosylation analysis [1] typically employs both single and tandem mass spectrometry (MS) to analyze digested glycoprotein samples at the level of intact glycopeptides. ...
Glycopeptides in peptide or digested protein samples pose a number of analytical and bioinformatics challenges beyond those posed by unmodified peptides or peptides with smaller posttranslational modifications. Exact structural elucidation of glycans is generally beyond the capability of a single mass spectrometry experiment, so a reasonable level of identification for tandem mass spectrometry, taken by several glycopeptide software tools, is that of peptide sequence and glycan composition, meaning the number of monosaccharides of each distinct mass, for example HexNAc(2)Hex(5) rather than man5. Even at this level, however, glycopeptide analysis poses challenges: finding glycopeptide spectra when they are a tiny fraction of the total spectra; assigning spectra with unanticipated glycans, not in the initial glycan database; and finding, scoring, and labeling diagnostic peaks in tandem mass spectra. Here we discuss recent improvements to Byonic, a glycoproteomics search program, that address these three issues. Byonic now supports filtering spectra by m/z peaks, so that the user can limit attention to spectra with diagnostic peaks, for example, at least two out of three of 204.087 for HexNAc, 274.092 for NeuAc (with water loss), and 366.139 for HexNAc-Hex, all within a set mass tolerance, for example, ± 0.01 Daltons. Also new is glycan "wildcard" search, which allows an unspecified mass within a user-set mass range to be applied to N- or O-linked glycans and enables assignment of spectra with unanticipated glycans. Finally the next release of Byonic supports user-specified peak annotations from user-defined posttranslational modifications. We demonstrate the utility of these new software features by finding previously unrecognized glycopeptides in publicly available data, including glycosylated neuropeptides from rat brain.
... in specific software such as Byonic, pGlyco, Mascot, Protein Prospector, sugar QB and others. 137,[186][187][188][189][190][191][192][193] Intact glycopeptide quantification can be performed by matching spectral features using MaxQuant and Proteome Discoverer (step [13][14]. 177,193,194 The identification of N-linked glycosylation sites in PNGase F-treated glycopeptides can be identified by the conversion of asparagine to aspartic acid signature during peptide sequencing. ...
Protein glycosylation is a co- and post-translational modification that, in Leishmania parasites, plays key roles in vector-parasite-vertebrate host interaction. In the mammalian host, Leishmania protein glycosylation is involved in virulence, host cell invasion, and immune evasion and modulation. The Leishmania glycocalyx is composed by a dense array of glycoconjugates including lipophosphoglycan, glycoinositolphospholipids, glycoproteins and proteophosphoglycans which varies in composition between Leishmania species and developmental stages. The current knowledge on Leishmania protein glycosylation is quite limited. The development of novel analytical tools to characterize the Leishmania glycoproteome and the expanding toolbox to modulate the parasite glycocode will help in deciphering the processes involved in Leishmania-host interaction. This review will recapitulate the current knowledge of Leishmania protein glycosylation, and glycan structures reported, and the potential application of mass spectrometry-based analysis for system-wide Leishmania glycoproteome and glycome analysis.
... Within this work we used open searching within Byonic, a widely used tool in the glycoproteomic community for the analysis of glycosylation (57,67,68). This enabled us to directly compare the performance of open searches to focused glycopeptide searches within the same platform. ...
Mass spectrometry has become an indispensable tool for the characterisation of glycosylation across biological systems. Our ability to generate rich fragmentation of glycopeptides has dramatically improved over the last decade yet our informatic approaches still lag behind. While glycoproteomic informatics approaches using glycan databases have attracted considerable attention, database independent approaches have not. This has significantly limited high throughput studies of unusual or atypical glycosylation events such as those observed in bacteria. As such, computational approaches to examine bacterial glycosylation and identify chemically diverse glycans are desperately needed. Here we describe the use of wide-tolerance (up to 2000 Da) open searching as a means to rapidly examine bacterial glycoproteomes. We benchmarked this approach using N-linked glycopeptides of Campylobacter fetus subsp. fetus as well as O-linked glycopeptides of Acinetobacter baumannii and Burkholderia cenocepacia revealing glycopeptides modified with a range of glycans can be readily identified without defining the glycan masses prior to database searching. Utilising this approach, we demonstrate how wide tolerance searching can be used to compare glycan utilisation across bacterial species by examining the glycoproteomes of eight Burkholderia species ( B. pseudomallei ; B. multivorans ; B. dolosa ; B. humptydooensis ; B. ubonensis , B. anthina ; B. diffusa ; B. pseudomultivorans ). Finally, we demonstrate how open searching enables the identification of low frequency glycoforms based on shared modified peptides sequences. Combined, these results show that open searching is a robust computational approach for the determination of glycan diversity within bacterial proteomes.
... The EMBO Journal 39: e103457 | 2020 lower one, suggesting that N-glycosylation of GluK2 and/or GluK3 may be impaired in SEZ6KO neurons ( Fig 4A). In fact, GluK2 and GluK3 have multiple N-glycosylation sites (Parker et al, 2013;Vernon et al, 2017), which are glycosylated to different extents, producing GluK2 and GluK3 forms with more simple, immature glycosylation and with mature glycosylation. GluK2/3 with immature glycans is mostly found within cells, whereas GluK2/3 with mature glycans localizes to a larger extent to the cell surface (Mah et al, 2005). ...
Seizure protein 6 (SEZ6) is required for the development and maintenance of the nervous system, is a major substrate of the protease BACE1 and is linked to Alzheimer's disease (AD) and psychiatric disorders, but its molecular functions are not well understood. Here, we demonstrate that SEZ6 controls glycosylation and cell surface localization of kainate receptors composed of GluK2/3 subunits. Loss of SEZ6 reduced surface levels of GluK2/3 in primary neurons and reduced kainate-evoked currents in CA1 pyramidal neurons in acute hippocampal slices. Mechanistically, loss of SEZ6 in vitro and in vivo prevented modification of GluK2/3 with the human natural killer-1 (HNK-1) glycan, a modulator of GluK2/3 function. SEZ6 interacted with GluK2 through its ectodomain and promoted post-endoplasmic reticulum transport of GluK2 in the secretory pathway in heterologous cells and primary neurons. Taken together, SEZ6 acts as a new trafficking factor for GluK2/3. This novel function may help to better understand the role of SEZ6 in neurologic and psychiatric diseases.
... CID performed using an Orbitrap equipped with HCD, also known as higher-energy C-trap dissociation has become a popular mode of fragmentation for glycopeptides. HCD spectra generally contain fragment ion intensities of both the glycosyl and peptidyl moieties of the parent ion (Parker et al., 2013;Nilsson, 2016). Oxonium ion ratios of these spectra have been associated with the glycan composition and enantiomeric specificity. ...
The advancement of viral glycomics has paralleled that of the mass spectrometry glycomics toolbox. In some regard the glycoproteins studied have provided the impetus for this advancement. Viral proteins are often highly glycosylated, especially those targeted by the host immune system. Glycosylation tends to be dynamic over time as viruses propagate in host populations leading to increased number of and/or “movement” of glycosylation sites in response to the immune system and other pressures. This relationship can lead to highly glycosylated, difficult to analyze glycoproteins that challenge the capabilities of modern mass spectrometry. In this review, we briefly discuss five general areas where glycosylation is important in the viral niche and how mass spectrometry has been used to reveal key information regarding structure–function relationships between viral glycoproteins and host cells. We describe the recent past and current glycomics toolbox used in these analyses and give examples of how the requirement to analyze these complex glycoproteins has provided the incentive for some advances seen in glycomics mass spectrometry. A general overview of viral glycomics, special cases, mass spectrometry methods and work‐flows, informatics and complementary chemical techniques currently used are discussed.
... Due to the important roles of protein glycosylation on the regulation of protein structures and functions, glycoproteomic analysis of HCC has been reported in several previous studies (26)(27)(28)(29). In these studies, the commonly used analytical strategy was to separate the glycans from the glycosylated peptides by enzymatic or chemical methods, and then separately performed mass spectrometry analysis on the released glycan or deglycosylated peptide. ...
Hepatocellular carcinoma (HCC) is still one of the malignant tumors with high morbidity and mortality in China and worldwide. Although alpha-fetoprotein (AFP) as well as core fucosylated AFP-L3 have been widely used as important biomarkers for HCC diagnosis and evaluation, the AFP level shows a huge variation among HCC patient populations. In addition, the AFP level has also been proved to be associated with pathological grade, progression, and survival of HCC patients. Understanding the intrinsic heterogeneities of HCC associated with AFP levels is essential for the molecular mechanism studies of HCC with different AFP levels as well as for the potential early diagnosis and personalized treatment of HCC with AFP negative. In this study, an integrated N-glycoproteomic and proteomic analysis of low and high AFP levels of HCC tumors was performed to investigate the intrinsic heterogeneities of site-specific glycosylation associated with different AFP levels of HCC. By large-scale profiling and quantifying more than 4,700 intact N-glycopeptides from 20 HCC and 20 paired paracancer samples, we identified many commonly altered site-specific N-glycans from HCC tumors regardless of AFP levels, including decreased modifications by oligo-mannose and sialylated bi-antennary glycans, and increased modifications by bisecting glycans. By relative quantifying the intact N-glycopeptides between low and high AFP tumor groups, the great heterogeneities of site-specific N-glycans between two groups of HCC tumors were also uncovered. We found that several sialylated but not core fucosylated tri-antennary glycans were uniquely increased in low AFP level of HCC tumors, while many core fucosylated bi-antennary or hybrid glycans as well as bisecting glycans were uniquely increased in high AFP tumors. The data provide a valuable resource for future HCC studies regarding the mechanism, heterogeneities and new biomarker discovery.
Human noroviruses, globally the main cause of viral gastroenteritis, show strain specific affinity for histo-blood group antigens (HBGA) and can successfully be propagated ex vivo in human intestinal enteroids (HIEs). HIEs established from jejunal stem cells of individuals with different ABO, Lewis and secretor geno- and phenotypes, show varying susceptibility to such infections. Using bottom-up glycoproteomic approaches we have defined and compared the N-linked glycans of glycoproteins of seven jejunal HIEs. Membrane proteins were extracted, trypsin digested, and glycopeptides enriched by hydrophilic interaction liquid chromatography and analyzed by nanoLC-MS/MS. The Byonic software was used for glycopeptide identification followed by hands-on verifications and interpretations. Glycan structures and attachment sites were identified from MS 2 spectra obtained by higher-energy collision dissociation through analysis of diagnostic saccharide oxonium ions (B-ions), stepwise glycosidic fragmentation of the glycans (Y-ions), and peptide sequence ions (b- and y-ions). Altogether 694 unique glycopeptides from 93 glycoproteins were identified. The N-glycans encompassed pauci- and oligomannose, hybrid- and complex-type structures. Notably, polyfucosylated HBGA-containing glycopeptides of the four glycoproteins tetraspanin-8, carcinoembryonic antigen-related cell adhesion molecule 5, sucrose-isomaltase and aminopeptidase N were especially prominent and were characterized in detail and related to donor ABO, Lewis and secretor types of each HIE. Virtually no sialylated N-glycans were identified for these glycoproteins suggesting that terminal sialylation was infrequent compared to fucosylation and HBGA biosynthesis. This approach gives unique site-specific information on the structural complexity of N-linked glycans of glycoproteins of human HIEs and provides a platform for future studies on the role of host glycoproteins in gastrointestinal infectious diseases.
Mass spectrometry-driven glycomics and glycoproteomics, the system-wide profiling of detached glycans and intact glycopeptides from biological samples, respectively, are powerful approaches to interrogate the heterogenous glycoproteome. Efforts to develop integrated workflows employing both glycomics and glycoproteomics have been invested since the concerted application of these complementary approaches enables a deeper exploration of the glycoproteome. This protocol paper outlines, step-by-step, an integrated -omics technology, the “glycomics-assisted glycoproteomics” method, that first establishes the N-glycan fine structures and their quantitative distribution pattern of protein extracts via porous graphitized carbon-LC-MS/MS. The N-glycome information is then used to augment and guide the challenging reversed-phase LC-MS/MS-based profiling of intact N-glycopeptides from the same protein samples. Experimental details and considerations relating to the sample preparation and the N-glycomics and N-glycoproteomics data collection, analysis, and integration are discussed. Benefits of the glycomics-assisted glycoproteomics method, which can be readily applied to both simple and complex biological specimens such as protein extracts from cells, tissues, and bodily fluids (e.g., serum), include quantitative information of the protein carriers and site(s) of glycosylation, site occupancy, and the site-specific glycan structures directly from biological samples. The glycomics-assisted glycoproteomics method therefore facilitates a comprehensive view of the complexity and dynamics of the heterogenous glycoproteome.
Neural communication and modulation are complex processes. Ionotropic glutamate receptors (iGluRs) significantly contribute to mediating the fast-excitatory branch of neurotransmission in the mammalian brain. Kainate receptors (KARs), a subfamily of the iGluRs, act as modulators of the neuronal circuitry by playing important roles at both the post- and presynaptic sites of specific neurons. The functional tetrameric receptors are formed by two different gene families, low agonist affinity (GluK1-GluK3) and high agonist affinity (GluK4-GluK5) subunits. These receptors garnered attention in the past three decades, and since then, much work has been done to understand their localization, interactome, physiological functions, and regulation. Cloning of the receptor subunits (GluK1-GluK5) in the early 1990s led to recombinant expression of kainate receptors in heterologous systems. This facilitated understanding of the functional differences between subunit combinations, splice variants, trafficking, and drug discovery. Structural studies of individual domains and recent full-length homomeric and heteromeric kainate receptors have revealed unique functional mechanisms, which have answered several long-standing questions in the field of kainate receptor biology. In this chapter, we review the current understanding of kainate receptors and associated disorders.
The composition of N-linked glycans that are conjugated to the prostate-specific membrane antigen (PSMA) and their functional significance in prostate cancer progression have not been fully characterized. PSMA was isolated from two metastatic prostate cancer cell lines, LNCaP and MDAPCa2b, which have different tissue tropism and localization. Isolated PSMA was trypsin-digested, and intact glycopeptides were subjected to LC-HCD-EThcD-MS/MS analysis on a Tribrid Orbitrap Fusion Lumos mass spectrometer. Differential qualitative and quantitative analysis of site-specific N-glycopeptides was performed using Byonic and Byologic software. Comparative quantitative analysis demonstrates that multiple glycopeptides at asparagine residues 51, 76, 121, 195, 336, 459, 476, and 638 were in significantly different abundance in the two cell lines (p < 0.05). Biochemical analysis using endoglycosidase treatment and lectin capture confirm the MS and site occupancy data. The data demonstrate the effectiveness of the strategy for comprehensive analysis of PSMA glycopeptides. This approach will form the basis of ongoing experiments to identify site-specific glycan changes in PSMA isolated from disease-stratified clinical samples to uncover targets that may be associated with disease progression and metastatic phenotypes.
For studies of glycan structure/function relationships of glycoconjugates, a detailed characterization of the carbohydrate moiety is a prerequisite. To this end, the glycans are usually released and isolated before analysis. This chapter provides protocols for the chemical and enzymatic release of different types of glycans from glycoconjugates and discusses the pro and cons of the different methods. A typical strategy for the analysis of glycoproteins is described. The preparation of glycopeptides and their isolation might form part of this process. Furthermore, a typical scheme for the analysis of proteoglycans and their glycosaminoglycans is provided. Also, attention is paid to the analysis of glycolipids and polysaccharides.
Background:
The aberrant proteolytic processing of amyloid precursor protein (APP) into amyloid β peptide (Aβ) in brain is a critical step in the pathogenesis of Alzheimer's disease (AD). As an O-glycosylated protein, O-glycosylation of APP is considered to be related to Aβ generation. Therefore, comprehensive analysis of APP O-glycosylation is important for understanding its functions.
Methods:
We developed a Targeted MS approach with Multi-Fragmentation techniques (TMMF strategy), and successfully characterized O-glycosylation profiling of APP695 expressed in HEK-293 T cells. We calculated relative abundance of glycopeptides with various O-glycosites and O-glycans, and further investigated the alteration of APP O-glycosylation upon TNF-α treatment.
Results:
A total of 14 O-glycosites were identified on three glycopeptides of APP, and at least four O-glycans including GalNAc (Tn antigen), core 1, and mono-/di-sialylated core 1 glycans were determinant at the residues of Thr576 and Thr577. We found a dense cluster of truncated O-glycans on the region nearby beginning of E2 domain and high abundance of sialylated O-glycans on the region close to β-cleavage site. Moreover, we also observed that TNF-α could upregulate the expression of APP and the truncated O-glycans on APP in HEK-293 T cell.
Conclusion:
Our study established an intact O-glycopeptide MS analysis strategy for APP O-glycopeptide identification with enhanced fragmentation efficiency and detection sensitivity. These results provide a comprehensive O-glycosylation map of APP expressed in HEK-293 T cell.
General significance:
The accurate O-glycosites and O-glycan structures on APP may lead to a better understanding of the roles O-glycosylation plays in the processing and functions of APP.
N-linked glycosylation is a ubiquitous protein modification that is capable of modulating protein structure, function and interactions. Many proteins in the brain associated with the synapse and important for synaptic transmission are highly glycosylated and their glycosylation could be important for learning and memory related molecular processes and synaptic plasticity. In the present study, we extend the knowledge of the synaptic glycome and glycoproteome by performing glycan- and intact glycopeptide-focused analyses of isolated rat nerve terminals (synaptosomes) by LC-MS/MS. Overall, glycomics identified a total of 41 N-glycans in isolated synaptosomes. Sialylated N-glycans represented only 7% of the total abundance of the rat synaptosome N-glycome with oligomannose, neutral hybrid and complex type N-glycans being the most abundant structures. Using detergent extraction of the active zone proteins from the synaptosomes revealed a change in the active zone glycan abundance in comparison with the rest of the synaptosome glycan content. Characterization of intact sialylated N-linked glycopeptides enriched by titanium dioxide chromatography revealed more than 85% selectivity of sialylated species and the presence of NeuGc on active zone proteins. In addition, both disialic and trisialic acid modified glycans were present on synaptic glycoproteins, although oxonium ion profiling revealed that trisialic units were only present on glycoproteins in the detergent soluble fraction. However, correct identification of intact sialylated N-linked glycopeptides using the Byonic program failed, most likely due to the lack of peptide backbone fragmentation during tandem mass spectrometry.
Glycans play a vital role in modulating protein structure and function from involvement in protein folding, solubility and stability to regulation of tissue distribution, recognition specificity, and biological activity. They can act as both positive and negative regulators of protein function, providing an additional level of control with respect to genetic and environmental conditions.
Due to the complexity of glycosylated protein forms, elucidating structural and functional information has been challenging task for researchers but recent development of chemical biology-based tools and techniques is bridging these knowledge gaps. This book provides a thorough review of the current state of glycoprotein chemical biology, describing the development and application of glycoprotein and glycan synthesis technologies for understanding and manipulating protein glycosylation.
Chinese hamster ovary (CHO) cells are one of the most commonly used host cell lines used for the production human therapeutic proteins. Much research over the past two decades has focussed on improving the growth, titre and cell specific productivity of CHO cells and in turn lowering the costs associated with production of recombinant proteins. CHO cell engineering has become of particular interest in recent years following the publication of the CHO cell genome and the availability of data relating to the proteome, transcriptome and metabolome of CHO cells. However, data relating to the cellular post-translational modification (PTMs) which can affect the functionality of CHO cellular proteins has only begun to be presented in recent years. PTMs are important to many cellular processes and can further alter proteins by increasing the complexity of proteins and their interactions. In this review, we describe the research presented from CHO cells to date related on three of the most important PTMs; glycosylation, phosphorylation and ubiquitination.
The heterogeneity and low abundance of protein glycosylation present challenging barriers to the analysis of intact glycopeptides, which is key to comprehensively understanding the role of glycosylation in an organism. Efficient and specific enrichment of intact glycopeptides could help greatly with this problem. Here, we propose a new enrichment strategy using a boronic acid (BA)-functionalized mesoporous graphene-silica composite (denoted as GO@mSiO2-GLYMO-APB) for isolating intact glycopeptides from complex biological samples. The merits of this composite, including high surface area and synergistic effect from size exclusion functionality of mesoporous material, hydrophilic interaction of silica, and the reversible covalent binding with BA, enable the effective and specific enrichment of both intact N- and O-glycopeptides. The results from the enrichment performance of the strategy evaluated by standard glycoproteins and the application to global N- and O-glycosylation analyses in human serum indicate the robustness and potential of the strategy for intact glycopeptide analysis.
Aim: The study sought to determine the patterns of N-glycan profiles among Type 2 diabetes mellitus (T2DM) patients over a 6-month period. Materials & methods: Biochemical and clinical data were obtained from 253 T2DM patients at baseline and follow-up. Ultra-performance liquid chromatography and statistical methods were applied for N-glycan profiling. Results: The coefficients of variation were 28% and 29% at baseline and follow-up, respectively, whereas the range of N-glycan variability was from 11% to 56%. Apart from GP1 (FA2) and GP29 (FA3G3S [3,3,3]3), the intra-individual variations of N-glycan peaks were not statistically significant. Conclusion: N-glycan profiles were stable over 6-month period in T2DM patients and could be used to monitor biochemical changes in relation with T2DM comorbidities.
Hydrophilic interaction liquid chromatography (HILIC) glycopeptide enrichment is an indispensable tool for the high-throughput characterization of glycoproteomes. Despite its utility, HILIC enrichment is associated with a number of shortcomings, including requiring large amounts of starting materials, potentially introducing chemical artifacts such as formylation when high concentrations of formic acid are used, and biasing/undersampling specific classes of glycopeptides. Here, we investigate HILIC enrichment-independent approaches for the study of bacterial glycoproteomes. Using three Burkholderia species (Burkholderia cenocepacia, Burkholderia Dolosa, and Burkholderia ubonensis), we demonstrate that short aliphatic O-linked glycopeptides are typically absent from HILIC enrichments, yet are readily identified in whole proteome samples. Using high-field asymmetric waveform ion mobility spectrometry (FAIMS) fractionation, we show that at high compensation voltages (CVs), short aliphatic glycopeptides can be enriched from complex samples, providing an alternative means to identify glycopeptide recalcitrant to hydrophilic-based enrichment. Combining whole proteome and FAIMS analyses, we show that the observable glycoproteome of these Burkholderia species is at least 25% larger than what was initially thought. Excitingly, the ability to enrich glycopeptides using FAIMS appears generally applicable, with the N-linked glycopeptides of Campylobacter fetus subsp. fetus also being enrichable at high FAIMS CVs. Taken together, these results demonstrate that FAIMS provides an alternative means to access glycopeptides and is a valuable tool for glycoproteomic analysis.
The complexity and dynamics of the immensely heterogeneous glycoproteome of the prostate cancer (PCa) tumour micro-environment remain incompletely mapped, a knowledge gap that impedes our molecular-level understanding of the disease. To this end, we have used sensitive glycomics and glycoproteomics to map the protein-, cell- and tumour grade-specific N- and O-glycosylation in surgically-removed PCa tissues spanning five histological grades (n = 10/grade) and tissues from patients with benign prostatic hyperplasia (n = 5). Quantitative glycomics revealed PCa grade-specific alterations of the oligomannosidic-, paucimannosidic- and branched sialylated complex-type N-glycans, and dynamic remodelling of the sialylated core 1- and core 2-type O-glycome. Deep quantitative glycoproteomics identified ~7,400 unique N-glycopeptides from 500 N-glycoproteins and ~500 unique O-glycopeptides from nearly 200 O-glycoproteins. With reference to a recent Tissue and Blood Atlas, our data indicate that paucimannosidic glycans of the PCa tissues arise mainly from immune cell-derived glycoproteins. Further, the grade-specific PCa glycosylation arises primarily from dynamics in the cellular makeup of the PCa tumour microenvironment across grades involving increased oligomannosylation of prostate-derived glycoproteins and decreased bisecting GlcNAcylation of N-glycans carried by the extracellular matrix proteins. Further, elevated expression of several oligosaccharyltransferase subunits and enhanced N-glycoprotein site occupancy were observed associated with PCa progression. Finally, correlations between the protein-specific glycosylation and PCa progression were observed including increased site-specific core 2-type O-glycosylation of collagen VI. In conclusion, integrated glycomics and glycoproteomics have enabled new insight into the complexity and dynamics of the tissue glycoproteome associated with PCa progression generating an important resource to explore the underpinning disease mechanisms.
Hydrophilic Interaction Liquid Chromatography (HILIC) glycopeptide enrichment is an indispensable tool for the high-throughput characterisation of glycoproteomes. Despite its utility, HILIC enrichment is associated with a number of short comings including requiring large amounts of starting material, potentially introducing chemical artefacts such as formylation, and biasing/under-sampling specific classes of glycopeptides. Here we investigate HILIC enrichment independent approaches for the study of bacterial glycoproteomes. Using three Burkholderia species (B. cenocepacia, B. dolosa and B. ubonensis) we demonstrate that short aliphatic O-linked glycopeptides are typically absent from HILIC enrichments yet are readily identified in whole proteome samples. Using Field Asymmetric Waveform IMS (FAIMS) fractionation we show that at low compensation voltages (CVs) short aliphatic glycopeptides can be enriched from complex samples providing an alternative means to identify glycopeptides recalcitrant to hydrophilic based enrichment. Combining whole proteome and FAIMS analysis we show that the observable glycoproteome of these Burkholderia species is at least 30% larger than initially thought. Excitingly, the ability to enrich glycopeptides using FAIMS appears generally applicable, with the N-linked glycopeptides of Campylobacter fetus subsp. fetus also enrichable at low FAIMS CVs. Taken together, these results demonstrate that FAIMS provides an alternative means to access glycopeptides and is a valuable tool for glycoproteomic analysis.
Many cell surface and secreted proteins are modified by the covalent addition of glycans that play an important role in the development of multicellular organisms. These glycan modifications enable communication between cells and the extracellular matrix via interactions with specific glycan-binding lectins and the regulation of receptor-mediated signaling. Aberrant protein glycosylation has been associated with the development of several muscular diseases suggesting essential glycan- and lectin-mediated functions in myogenesis and muscle development but our molecular understanding of the precise glycans, catalytic enzymes and lectins involved remain only partially understood. Here, we quantified dynamic remodeling of the membrane-associated proteome during a time-course of myogenesis in cell culture. We observed wide-spread changes in the abundance of several important lectins and enzymes facilitating glycan biosynthesis. Glycomics-based quantification of released N-linked glycans confirmed remodeling of the glycome consistent with the regulation of glycosyltransferases and glycosidases responsible for their formation including a previously unknown di-galactose-to-sialic acid switch supporting a functional role of these glycoepitopes in myogenesis. Furthermore, dynamic quantitative glycoproteomic analysis with multiplexed stable isotope labelling and analysis of enriched glycopeptides with multiple fragmentation approaches identified glycoproteins modified by these regulated glycans including several integrins and growth factor receptors. Myogenesis was also associated with the regulation of several lectins most notably the up-regulation of galectin-1 (LGALS1). CRISPR/Cas9-mediated deletion of Lgals1 inhibited differentiation and myotube formation suggesting an early functional role of galectin-1 in the myogenic program. Importantly, similar changes in N-glycosylation and the up-regulation of galectin-1 during postnatal skeletal muscle development were observed in mice. Treatment of new-born mice with recombinant adeno-associated viruses to overexpress galectin-1 in the musculature resulted in enhanced muscle mass. Our data form a valuable resource to further understand the glycobiology of myogenesis and will aid the development of intervention strategies to promote healthy muscle development or regeneration.
Site-specific characterization of glycosylation requires intact glycopeptide analysis, and recent efforts have focused on how to best interrogate glycopeptides using tandem mass spectrometry (MS/MS). Beam-type collisional activation, i.e., higher-energy collisional dissociation (HCD), has been a valuable approach, but stepped collision energy HCD (sceHCD) and electron transfer dissociation with HCD supplemental activation (EThcD) have emerged as potentially more suitable alternatives. Both sceHCD and EThcD have been used with success in large-scale glycoproteomic experiments, but they each incur some degree of compromise. Most progress has occurred in the area N-glycoproteomics. There is growing interest in extending this progress to O-glycoproteomics, which necessitates comparisons of method performance for the two classes of glycopeptides. Here, we systematically explore the advantages and disadvantages of conventional HCD, sceHCD, ETD, and EThcD for intact glycopeptide analysis and determine their suitability for both N- and O-glycoproteomic applications. For N-glycopeptides, HCD and sceHCD generate similar numbers of identifications, although sceHCD generally provides higher quality spectra. Both significantly outperform EThcD methods, indicating that ETD-based methods are not required for routine N-glycoproteomics. Conversely, ETD-based methods, especially EThcD, are indispensable for site-specific analyses of O-glycopeptides. Our data show that O-glycopeptides cannot be robustly characterized with HCD-centric methods that are sufficient for N-glycopeptides, and glycoproteomic methods aiming to characterize O-glycopeptides must be constructed accordingly.
N‐glycans from glycoproteins are complex, branched structures whose structural determination presents many analytical problems. Mass spectrometry, usually conducted in positive ion mode, often requires extensive sample manipulation, usually by derivatization such as permethylation, to provide the necessary structure‐revealing fragment ions. The newer but, so far, lesser used negative ion techniques, on the contrary, provide a wealth of structural information not present in positive ion spectra that greatly simplify the analysis of these compounds and can usually be conducted without the need for derivatization. This review describes the use of negative ion mass spectrometry for the structural analysis of N‐linked glycans and emphasises the many advantages that can be gained by this mode of operation. Biosynthesis and structures of the compounds are described followed by methods for release of the glycans from the protein. Methods for ionization are discussed with emphasis on matrix‐assisted laser desorption/ionization (MALDI) and methods for producing negative ions from neutral compounds. Acidic glycans naturally give deprotonated species under most ionization conditions. Fragmentation of negative ions is discussed next with particular reference to those ions that are diagnostic for specific features such as the branching topology of the glycans and substitution positions of moieties such as fucose and sulfate, features that are often difficult to identify easily by conventional techniques such as positive ion fragmentation and exoglycosidase digestions. The advantages of negative over positive ions for this structural work are emphasised with an example of a series of glycans where all other methods failed to produce a structure. Fragmentation of derivatized glycans is discussed next, both with respect to derivatives at the reducing terminus of the molecules, and to methods for neutralization of the acidic groups on sialic acids to both stabilize them for MALDI analysis and to produce the diagnostic fragments seen with the neutral glycans. The use of ion mobility, combined with conventional mass spectrometry is described with emphasis on its use to extract clean glycan spectra both before and after fragmentation, to separate isomers and its use to extract additional information from separated fragment ions. A section on applications follows with examples of the identification of novel structures from lower organisms and tables listing the use of negative ions for structural identification of specific glycoproteins, glycans from viruses and uses in the biopharmaceutical industry and in medicine. The review concludes with a summary of the advantages and disadvantages of the technique.
Precise assignment of sialylation linkages at the glycopeptide level is of importance in bottom-up glycoproteomics and an indispensable step to understand the function of glycoproteins in pathogen-host interactions and cancer progression. Even though some efforts have been dedicated to the discrimination of α2,3/α2,6-sialylated isomers, unambiguous identification of sialoglycopeptide isomers is still needed. Herein, we developed an innovative glycosyltransferase labeling assisted mass spectrometry (GLAMS) strategy. After specific enzymatic labeling, oxonium ions from higher-energy C-trap dissociation (HCD) fragmentation of α2,3-sailoglycopeptides then generate unique reporters to distinctly differentiate those of α2,6-sailoglycopeptide isomers. With this strategy, a total of 1,236 linkage-specific sialoglycopeptides were successfully identified from 161 glycoproteins in human serum.
Intact glycopeptide analysis is becoming more common with developments in mass spectrometry instrumentation and fragmentation approaches. In particular, collision-based fragmentation approaches such as higher energy collisional dissociation (HCD) and radical-driven fragmentation approaches such as electron transfer dissociation (ETD) provide complementary information, but bioinformatic strategies to utilize this combined information are currently lacking. In this work we adapted a software tool, MS-Filter, to search HCD peak list files for predicted Y ions based on matched EThcD results to propose additional glycopeptide assignments. The strategy proved to be extremely powerful for O-glycopeptide data, and also of benefit for N-linked data, where it allowed rescue of low confidence results from database searching.
Glycopeptides represent cross-linked structures between chemically and physically different biomolecules. Mass spectrometric analysis of O-glycopeptides may reveal the identity of the peptide, the composition of the glycan and even the connection between certain sugar units, but usually only the combination of different MS/MS techniques provides sufficient information for reliable assignment. Currently, HCD analysis followed by diagnostic sugar fragment-triggered ETD or EThcD experiments is the most promising data acquisition protocol. However, the information content of the different MS/MS data is handled separately by search engines. We are convinced that these data should be used in concert, as we demonstrate in the present study. First, glycopeptides bearing the most common glycans can be identified from EThcD and/or HCD data. Then, searching for Y0 (the gas-phase deglycosylated peptide) in HCD spectra, the potential glycoforms of these glycopeptides could be lined up. Finally, these spectra and the corresponding EThcD data can be used to verify or discard the tentative assignments and to obtain further structural information about the glycans. We present 18 novel human urinary sialoglycan structures deciphered using this approach. To accomplish this in an automated fashion further software development is necessary.
Backgroud:
Given the increasing morbidity and mortality of colorectal cancer (CRC), it is urgent to develop a noninvasive screening strategy for early diagnosis of CRC. Altered IgG glycosylation is associated with CRC progression, whereas the association of IgG isomeric glycosylation with CRC were not investigated.
Methods:
Methylamidation of IgG N-glycans was conducted prior to PGC-based nanoLC-ESI-MS/MS analysis. Data processing was operated by a self-developed application based on MATLAB solution. Statistical analysis including K-S test, t-test, ROC curve and OPLS-DA were successively performed. Additionally, an independent set was utilized to validate the results.
Results:
Total 28 IgG glycans and 79 compositional isomers were identified, over half of which are firstly identified so far. Statistical analysis showed that CRC associates with increase in IgG agalactosylation, decrease in IgG sialylation and fucosylation of sialylated glycans. Additionally, it was found that three compositional isomers (H3N4F1-a, H3N4F1-b and H4N3S1F1-e) could distinguish CRC and early stages from controls with an accurate area under the receiver operating characteristic curve. Significantly, these results were validated in an independent set by multivariate statistical analysis.
Conclusions:
This is the first comprehensively profiling of isomer-specific IgG N-glycosylation, which could differentiate normal controls from colorectal disease patients. The candidate IgG glyco-biomarkers provide important screening indicators for early diagnosis of CRC.
General significance:
Colorectal cancer progression is strongly associated with isomer-specific IgG N-glycosylation.
Crustaceans are commonly used model organisms to study neuromodulation. Despite numerous reported crustacean neuropeptide families and their functions, there has been no report on neuropeptide glycosylation. This is in part due to a lack of sensitive method that enables deciphering this intricate low-abundance post-translational modification (PTM), even though glycosylation has been shown to play an important role in neuromodulation. Here, we describe the discovery of glycosylated neuropeptides with an enrichment-free approach taking advantage of signature oxonium ions produced in higher-energy collision dissociation (HCD) MS/MS spectra. The detection of the oxonium ions in the HCD scans suggests glycan attachment to peptides, allowing electron-transfer/higher-energy collision dissociation (EThcD) to be performed to selectively elucidate structural information of glycosylated neuropeptides that are buried in non-glycosylated peptides. Overall, 4 N-linked and 14 O-linked glycosylated neuropeptides have been identified for the first time in the crustacean nervous system. In addition, 91 novel putative neuropeptides have been discovered based on the collected HCD scans. This hybrid approach, coupling shotgun method for neuropeptide discovery and targeted strategy for glycosylation characterization enables the first report on glycosylated neuropeptides in crustacean and the discovery of additional neuropeptides simultaneously. The elucidation of novel glycosylated neuropeptides sheds light on the crustacean peptidome and offers novel insights into future neuropeptide functional studies.
Glycosylation is a common protein PTM, and its aberrant regulation has been widely linked to various pathological conditions including cancers. Our recent development of intact N-glycopeptide search engine GPSeeker has enabled relatively quantitative structure-specific characterization of differentially expressed intact N-glycopeptides (DEGPs) with isotopic labeling of the peptide backbones and structure-specific fragment ions of the N-glycan moieties. Here we report our site- and structure-specific relatively quantitative N-glycoproteomics study of breast MCF-7 cancer cells (relative to epithelial MCF-10A cells) using RPLC-nanoESI-MS/MS and GPSeeker. With spectrum-level FDR ≤ 1%, 581 intact N-glycopeptides with comprehensive structural information of both the peptide backbones (amino acid sequences, N-glycosites) and the N-glycan moieties (monosaccharide composition, sequence and linkages) were identified from five technical replicates (TR1, TR2, TR3, TR4 and TR5). With the criteria of quantified at least thrice out of the five technical replicates with no <1.5-fold change, p ≤ .05 and RSD ≤ 20%, 56 DEGPs were quantified from 23 N-glycosites on 19 intact N-glycoproteins. For the 19 intact N-glycoproteins observed with differential N-glycosylation expression, 14 (each with one or more DEGPs) were observed with uniform down regulation; 6 (each with one or more DEGPs) were observed with uniform up regulation; whereas one was observed with both up and down regulation.
Significance
Differential N-glycosylation in breast MCF-7 cancer cells (relative to MCF-10A epithelial cells) were qualitatively and quantitatively characterized with site- and structure-specific N-glycoproteomics using RPLC-nanoESI-MS/MS (HCD with stepped NCEs) and intact N-glycopeptide search engine GPSeeker. With spectrum-level FDR ≤ 1%, 581 intact N-glycopeptides with comprehensive structural information of both the peptide backbones and the N-glycan moieties were identified; For the 248 putative N-glycosites, 248 were confirmed where 125 have not been annotated in UniProt as of July 25, 2019. For the 114 N-glycan putative linkage structures, 44 were confirmed with no less than one structure-diagnostic fragment ions. With the criteria of quantified at least thrice out of the five technical replicates with no < 1.5-fold change and p ≤ .05, 56 DEGPs were quantified from 21 intact N-glycoproteins; 13 and 5 intact N-glycoproteins (each with one or more DEGPs) were observed with uniform down and up regulation; whereas one were observed with simultaneous up and down regulation.
We present the first large scale study characterizing both N- and O-linked glycosylation in a site-specific manner on hundreds of proteins. We demonstrate that a lectin-affinity fractionation step utilizing wheat germ agglutinin enriches not only peptides carrying intracellular O-GlcNAc, but also those bearing ER/Golgi-derived N- and O-linked carbohydrate structures. LC/MS analysis with high accuracy precursor mass measurements and high sensitivity ion trap electron-transfer dissociation (ETD) were utilized for structural characterization of glycopeptides. Our results reveal both the identity of the precise sites of glycosylation and information on the oligosaccharide structures possible on these proteins. We report a novel iterative approach that allowed us to interpret the ETD dataset directly without making prior assumptions about the nature and distribution of oligosaccharides present in our glycopeptide mixture. Over 2500 unique N- and O-linked glycopeptides were identified on 453 proteins. The extent of microheterogeneity varied extensively, and up to 19 different oligosaccharides were attached at a given site. We describe the presence of the well-known mucin-type structures for O-glycosylation, an EGF-domain-specific fucosylation and a rare O-mannosylation on the transmembrane phosphatase Ptprz1. Finally, we identified three examples of O-glycosylation on tyrosine residues.
Author Summary
This study was performed to determine what possible glycan receptors for influenza were present in the human respiratory tract. We compared the glycans present on existing published glycan arrays with the actual glycans identified in the human respiratory tract by mass spectrometric analysis to determine how representative these arrays would be for potential binding. The most comprehensive array to date only contained approximately half the range of the actual glycans present. Over the past 5 years we have performed ex-vivo infection of 113 bronchial and 185 lung samples with seasonal, avian and swine influenza viruses, and have demonstrated that the lung is able to be infected by all types of influenza viruses but that the bronchus can also be infected by a limited range of avian, swine and seasonal viruses. The key findings are that there is wide spectrum of glycans present in the respiratory tract which can be used by influenza viruses for infection, and the currently available arrays are not predictive of successful infection. Our findings will be of use for researchers in developing more comprehensive and focused arrays for the screening of emerging influenza viruses and bacteria in order to determine their potential threat to humans.
Previous proteomics studies have partially unravelled the complexity of endothelial protein secretion, but did not investigate glycosylation, a key modification on secreted and membrane proteins for cell communication. In this study, human umbilical vein endothelial cells were kept in serum-free medium before activation by phorbol 12-myristate 13-acetate, a commonly used secretagogue that induces exocytosis of endothelial vesicles. Apart from 123 secreted proteins, the secretome was particularly rich in membrane proteins. Glycopeptides were enriched by ZIC-HILIC resins and were either treated with PNGase F and H(2)(18)O or directly analysed using a recently developed HCD-ETD workflow for a hybrid linear ion trap-orbitrap MS. After deglycosylation with PNGase F in the presence of H(2)(18)O, 123 unique peptides displayed (18)O-deamidation of asparagine, corresponding to 86 proteins with a total of 121 glycosylation sites. Direct glycopeptides analysis by HCD-ETD identified 131 glycopeptides from 59 proteins and 118 glycosylation sites, of which 41 were known, 51 were predicted and 26 were novel. Two methods were compared: alternating HCD-ETD and HCD-product dependent ETD. The former detected predominantly high intensity, multiply charged glycopeptides while the latter preferentially selected precursors with complex / hybrid glycans for fragmentation. Validation was performed by glycoprotein enrichment and analysing the input, the flow through and the bound fraction. This study represents the most comprehensive characterisation of endothelial protein secretion to date and demonstrates the potential of new HCD-ETD workflows for determining the glycosylation status in complex biological samples.
Byonic is the name of a software package for peptide and protein identification by tandem mass spectrometry. This software, which has only recently become commercially available, facilitates a much wider range of search possibilities than previous search software such as SEQUEST and Mascot. Byonic allows the user to define an essentially unlimited number of variable modification types. Byonic also allows the user to set a separate limit on the number of occurrences of each modification type, so that a search may consider only one or two chance modifications such as oxidations and deamidations per peptide, yet allow three or four biological modifications such as phosphorylations, which tend to cluster together. Hence, Byonic can search for tens or even hundreds of modification types simultaneously without a prohibitively large combinatorial explosion. Byonic's Wildcard Search allows the user to search for unanticipated or even unknown modifications alongside known modifications. Finally, Byonic's Glycopeptide Search allows the user to identify glycopeptides without prior knowledge of glycan masses or glycosylation sites. Curr. Protoc. Bioinform. 40:13.20.1-13.20.14. © 2012 by John Wiley & Sons, Inc.
An important step in mass spectrometry (MS)-based proteomics is the identification of peptides by their fragment spectra. Regardless of the identification score achieved, almost all MS/MS spectra contain remaining peaks that are not assigned by the search engine. These peaks may be explainable by human experts but the scale of modern proteomics experiments makes this impractical. In computer science, expert systems are a mature technology to implement a list of rules generated by interviews with practitioners. We here develop such an expert system, making use of literature knowledge as well as a large body of high mass accuracy and pure fragmentation spectra. Interestingly, we find that even with high mass accuracy data, rule sets can quickly become too complex, leading to over-annotation. Therefore we establish a rigorous false discovery rate, calculated by random insertion of peaks from a large collection of other MS/MS spectra, and use it to develop an optimized knowledge base. This rule set correctly annotates almost all peaks of medium or high abundance. For high resolution HCD data, median intensity coverage of fragment peaks in MS/MS spectra increases from 58% by search engine annotation alone to 86%. The resulting annotation performance surpasses a human expert, especially on complex spectra such as those of larger phosphorylated peptides. Our system is also applicable to high resolution CID data. It is available both as a part of MaxQuant and via a webserver that only requires an MS/MS spectrum and the corresponding peptides sequence, and which outputs publication quality, annotated MS/MS spectra (www.biochem.mpg.de/mann/tools/). It provides expert knowledge to beginners in the field of MS-based proteomics and helps expert users to focus on unusual and possibly novel types of fragment ions.
Selected reaction monitoring (SRM) on a triple quadrupole (QqQ) mass spectrometer is currently experiencing a renaissance within the proteomics community for its, as yet, unparalleled ability to characterize and quantify a set of proteins reproducibly, completely, and with high sensitivity. Given the immense benefit that high resolution and accurate mass (HR/AM) instruments have brought to the discovery proteomics field, we wondered if highly accurate mass measurement capabilities could be leveraged to provide benefits in the targeted proteomics domain as well. Here, we propose a new targeted proteomics paradigm centered on the use of next generation, quadrupole-equipped HR/AM instruments: parallel reaction monitoring (PRM). In PRM, the third quadrupole of a QqQ is substituted with a HR/AM mass analyzer to permit the parallel detection of all target product ions in one, concerted high resolution mass analysis. We detail the analytical performance of the PRM method, using a quadrupole-equipped bench-top Orbitrap MS, and draw a performance comparison to SRM in terms of run-to-run reproducibility, dynamic range, and measurement accuracy. In addition to requiring minimal upfront method development and facilitating automated data analysis, PRM yielded quantitative data over a wider dynamic range than SRM in the presence of a yeast background matrix due to PRM's high selectivity in the mass-to-charge domain. With achievable linearity over the quantifiable dynamic range found to be statistically equal between the two methods, our investigation suggests that PRM will be a promising new addition to the quantitative proteomics toolbox.
Growing evidence indicates that the individualized and highly reproducible N-glycan repertoires on each protein glycosylation site modulate function. Relationships between protein structures and the resulting N-glycoforms have been previously observed, but remain to be quantitatively confirmed and examined in detail to define the responsible mechanisms in the conserved mammalian glycosylation machinery. Here, we investigate this relationship by manually extracting and analyzing quantitative and qualitative site-specific glycoprofiling data from 117 research papers. Specifically, N-glycan structural motifs were correlated with the structure of the protein carriers, focusing on the solvent accessibility of the individual glycosylation sites and the physicochemical properties of the surrounding polypeptide chains. In total, 474 glycosylation sites from 169 mammalian N-glycoproteins originating from different tissues/body fluids were investigated. It was confirmed statistically that the N-glycan type, degree of core fucosylation and branching are strongly influenced by the glycosylation site accessibility. For these three N-glycan features, glycosylation sites carrying highly processed glycans were significantly more solvent accessible than those carrying less processed counterparts. The glycosylation site accessibilities could be linked to molecular signatures at the primary and secondary protein level, most notably to the glycoprotein size and the proportion of glycosylation sites located in accessible β-turns. In addition, the subcellular location of the glycoproteins influenced the formation of the N-glycan structures. These data confirm that protein structures dictate site-specific formation of several features of N-glycan structures by affecting the biosynthetic pathway. Mammals have, as such, evolved mechanisms enabling proteins to influence the N-glycans they present to the extracellular environment.
This protocol shows how to obtain a detailed glycan compositional and structural profile from purified glycoproteins or protein mixtures, and it can be used to distinguish different isobaric glycan isomers. Glycoproteins are immobilized on PVDF membranes before the N-glycans are enzymatically released by PNGase F, isolated and reduced. Subsequently, O-glycans are chemically released from the same protein spot by reductive β-elimination. After desalting with cation exchange microcolumns, the glycans are separated and analyzed by porous graphitized carbon liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Optionally, the glycans can be treated with sialidases or other specific exoglycosidases to yield more detailed structural information. The sample preparation takes approximately 4 d, with a heavier workload on days 2 and 3, and a lighter load on days 1 and 4. The time for data interpretation depends on the complexity of the samples analyzed. This method can be used in conjunction with the analysis of enriched glycopeptides by capillary/nanoLC-ESI-MS/MS, which together provide detailed information regarding the site heterogeneity of glycosylation.
Two different workflows were tested in order to develop methods that provide deeper insight into the secreted O-glycoproteome. Bovine serum samples were subjected to lectin affinity-chromatography both at the protein- and peptide-level in order to selectively isolate glycopeptides with the most common, mucin core-1 sugar. This enrichment step was implemented with either protein-level mixed-bed ion-exchange chromatography or with peptide-level electrostatic repulsion hydrophilic interaction chromatography. Both methods led to at least 65% of the identified products being glycopeptides, in comparison to ≈ 25% without the additional chromatography steps [Darula, Z., and Medzihradszky, K. F. (2009) Affinity enrichment and characterization of mucin core-1 type glycopeptides from bovine serum. Mol. Cell. Proteomics 8, 2515-2526]. In order to improve not only the isolation but also the characterization of the glycopeptides exoglycosidases were used to eliminate carbohydrate extensions from the directly peptide-bound GalNAc units. Consequent tandem MS analysis of the mixtures using higher-energy collision-dissociation and electron-transfer dissociation led to the identification of 124 glycosylation sites in 51 proteins. While the electron-transfer dissociation data provided the bulk of the information for both modified sequence and modification site assignment, the higher-energy collision-dissociation data frequently yielded confirmation of the peptide identity, and revealed the presence of some core-2 or core-3 oligosaccharides. More than two-thirds of the sites as well as the proteins have never been reported modified.
Urine is a complex mixture of proteins and waste products and a challenging biological fluid for biomarker discovery. Previous proteomic studies have identified more than 2800 urinary proteins but analyses aimed at unraveling glycan structures and glycosylation sites of urinary glycoproteins are lacking. Glycoproteomic characterization remains difficult because of the complexity of glycan structures found mainly on asparagine (N-linked) or serine/threonine (O-linked) residues. We have developed a glycoproteomic approach that combines efficient purification of urinary glycoproteins/glycopeptides with complementary MS-fragmentation techniques for glycopeptide analysis. Starting from clinical sample size, we eliminated interfering urinary compounds by dialysis and concentrated the purified urinary proteins by lyophilization. Sialylated urinary glycoproteins were conjugated to a solid support by hydrazide chemistry and trypsin digested. Desialylated glycopeptides, released through mild acid hydrolysis, were characterized by tandem MS experiments utilizing collision induced dissociation (CID) and electron capture dissociation fragmentation techniques. In CID-MS(2), Hex(5)HexNAc(4)-N-Asn and HexHexNAc-O-Ser/Thr were typically observed, in agreement with known N-linked biantennary complex-type and O-linked core 1-like structures, respectively. Additional glycoforms for specific N- and O-linked glycopeptides were also identified, e.g. tetra-antennary N-glycans and fucosylated core 2-like O-glycans. Subsequent CID-MS(3), of selected fragment-ions from the CID-MS(2) analysis, generated peptide specific b- and y-ions that were used for peptide identification. In total, 58 N- and 63 O-linked glycopeptides from 53 glycoproteins were characterized with respect to glycan- and peptide sequences. The combination of CID and electron capture dissociation techniques allowed for the exact identification of Ser/Thr attachment site(s) for 40 of 57 putative O-glycosylation sites. We defined 29 O-glycosylation sites which have, to our knowledge, not been previously reported. This is the first study of human urinary glycoproteins where "intact" glycopeptides were studied, i.e. the presence of glycans and their attachment sites were proven without doubt.
Zinc-finger nuclease (ZFN) gene targeting is emerging as a versatile tool for engineering of multiallelic gene deficiencies. A longstanding obstacle for detailed analysis of glycoproteomes has been the extensive heterogeneities in glycan structures and attachment sites. Here we applied ZFN targeting to truncate the O-glycan elongation pathway in human cells, generating stable 'SimpleCell' lines with homogenous O-glycosylation. Three SimpleCell lines expressing only truncated GalNAcα or NeuAcα2-6GalNAcα O-glycans were produced, allowing straightforward isolation and sequencing of GalNAc O-glycopeptides from total cell lysates using lectin chromatography and nanoflow liquid chromatography-mass spectrometry (nLC-MS/MS) with electron transfer dissociation fragmentation. We identified >100 O-glycoproteins with >350 O-glycan sites (the great majority previously unidentified), including a GalNAc O-glycan linkage to a tyrosine residue. The SimpleCell method should facilitate analyses of important functions of protein glycosylation. The strategy is also applicable to other O-glycoproteomes.
Resistance to tubulin-binding agents used in cancer is often multifactorial and can include changes in drug accumulation and modified expression of tubulin isotypes. Glycans on cell membrane proteins play important roles in many cellular processes such as recognition and apoptosis, and this study investigated whether changes to the glycan structures on cell membrane proteins occur when cells become resistant to drugs. Specifically, we investigated the alteration of glycan structures on the cell membrane proteins of human T-cell acute lymphoblastic leukemia (CEM) cells that were selected for resistance to desoxyepothilone B (CEM/dEpoB). The glycan profile of the cell membrane glycoproteins was obtained by sequential release of N- and O-glycans from cell membrane fraction dotted onto polyvinylidene difluoride membrane with PNGase F and β-elimination respectively. The released glycan alditols were analyzed by liquid chromatography (graphitized carbon)-electrospray ionization tandem MS. The major N-glycan on CEM cell was the core fucosylated α2-6 monosialo-biantennary structure. Resistant CEM/dEpoB cells had a significant decrease of α2-6 linked sialic acid on N-glycans. The lower α2-6 sialylation was caused by a decrease in activity of β-galactoside α2-6 sialyltransferase (ST6Gal), and decreased expression of the mRNA. It is clear that the membrane glycosylation of leukemia cells changes during acquired resistance to dEpoB drugs and that this change occurs globally on all cell membrane glycoproteins. This is the first identification of a specific glycan modification on the surface of drug resistant cells and the mechanism of this downstream effect on microtubule targeting drugs may offer a route to new interventions to overcome drug resistance.
Identification of deamidated sites in proteins is commonly used for assignment of N-glycosylation sites. It is also important for assessing the role of deamidation in vivo. However, nonenzymatic deamidation occurs easily in peptides under conditions commonly used in treatment with trypsin and PNGase F. The impact on proteomic sample preparation has not yet been evaluated systematically. In addition, the (13)C peaks of amidated peptides can be misassigned as monoisotopic peaks of the corresponding deamidated ones in database searches. The 19.34 mDa mass difference between them is proposed as a means for eliminating the resulting false positive identifications in large-scale proteomic analysis. We evaluated five groups of proteomic data, obtained mainly through an electrostatic repulsion-hydrophilic interaction chromatography (ERLIC)-reverse phase (RP) chromatography sequence, and ascertained that nonenzymatic asparagine deamidation occurred to some extent on 4-9% of the peptides, resulting in the false positive identification of many N-glycosylation sites. A comprehensive investigation indicated that the chief causative factors were the mildly alkaline pH and prolonged incubations at 37 °C during proteomic sample preparation. An improved protocol is proposed featuring tryptic digestion at pH 6 and deglycosylation at pH 5, resulting in a significant decrease in nonenzymatic deamidation while conserving adequate digestion efficiency. The number of identified deamidation sites was improved significantly by increasing the sample loading amount in liquid chromatography-tandem MS. This permitted the identification of a significant number of glutamine deamidation sites, which featured sequence motifs largely different from those for asparagine deamidation: -Q-V-, -Q-L- and -Q-G- and, to a lesser extent, -Q-A- and -Q-E-.
Protein glycosylation is an important posttranslational process, which regulates protein folding and functional expression. Studies have shown that abnormal glycosylation in tumor cells affects cancer progression and malignancy. In the current study, we have identified sialylated proteins using an alkynyl sugar probe in two different lung cancer cell lines, CL1-0 and CL1-5 with distinct invasiveness derived from the same parental cell line. Among the identified sialylated proteins, epidermal growth factor receptor (EGFR) was chosen to understand the effect of sialylation on its function. We have determined the differences in glycan sequences of EGFR in both cells and observed higher sialylation and fucosylation of EGFR in CL1-5 than in CL1-0. Further study suggested that overexpression of sialyltransferases in CL1-5 and α1,3-fucosyltransferases (FUT4 or FUT6) in CL1-5 and A549 cells would suppress EGFR dimerization and phosphorylation upon EGF treatment, as compared to the control and CL1-0 cells. Such modulating effects on EGFR dimerization were further confirmed by sialidase or fucosidase treatment. Thus, increasing sialylation and fucosylation could attenuate EGFR-mediated invasion of lung cancer cells. However, incorporation of the core fucose by α1,6-fucosylatransferase (FUT8) would promote EGFR dimerization and phosphorylation.
Human corticosteroid-binding globulin (CBG), a heavily glycosylated protein containing six N-linked glycosylation sites, transports cortisol and other corticosteroids in blood circulation. Here, we investigate the biological importance of the N-glycans of CBG derived from human serum by performing a structural and functional characterization of CBG N-glycosylation. Liquid chromatography-tandem MS-based glycoproteomics and glycomics combined with exoglycosidase treatment revealed 26 complex type N-glycoforms, all of which were terminated with α2,3-linked neuraminic acid (NeuAc) residues. The CBG N-glycans showed predominantly bi- and tri-antennary branching, but higher branching was also observed. N-glycans from all six N-glycosylation sites were identified with high site occupancies (70.5-99.5%) and glycoforms from all sites contained a relatively low degree of core-fucosylation (0-34.9%). CBG showed site-specific glycosylation and the site-to-site differences in core-fucosylation and branching could be in silico correlated with the accessibility to the individual glycosylation sites on the maturely folded protein. Deglycosylated and desialylated CBG analogs were generated to investigate the biological importance of CBG N-glycans. As a functional assay, MCF-7 cells were challenged with native and glycan-modified CBG and the amount of cAMP, which is produced as a quantitative response upon CBG binding to its cell surface receptor, was used to evaluate the CBG:receptor interaction. The removal of both CBG N-glycans and NeuAc residues increased the production of cAMP significantly. This confirms that N-glycans are involved in the CBG:receptor interaction and indicates that the modulation is performed by steric and/or electrostatic means through the terminal NeuAc residues.
The Sulfs are a family of endosulfatases that selectively modify the 6O-sulfation state of cell-surface heparan sulfate (HS) molecules. Sulfs serve as modulators of cell-signaling events because the changes they induce alter the cell surface co-receptor functions of HS chains. A variety of studies have been aimed at understanding how Sulfs modify HS structure, and many of these studies utilize Sulf knockout cell lines as the source for the HS used in the experiments. However, genetic manipulation of Sulfs has been shown to alter the expression levels of HS biosynthetic enzymes, and in these cases an assessment of the fine structural changes induced solely by Sulf enzymatic activity is not possible. Therefore, the present work aims to extend the understanding of substrate specificities of HSulf2 using in vitro experiments to compare HSulf2 activities on HS from different organ tissues.
To further the understanding of Sulf enzymatic activity, we conducted in vitro experiments where a variety of mammalian HS substrates were modified by recombinant human Sulf2 (HSulf2). Subsequent to treatment with HSulf2, the HS samples were exhaustively depolymerized and analyzed using size-exclusion liquid chromatography-mass spectrometry (SEC-LC/MS). We found that HSulf2 activity was highly dependent on the structural features of the HS substrate. Additionally, we characterized, for the first time, the activity of HSulf2 on the non-reducing end (NRE) of HS chains. The results indicate that the action pattern of HSulf2 at the NRE is different compared to internally within the HS chain.
The results of the present study indicate that the activity of Sulfs is dependent on the unique structural features of the HS populations that they edit. The activity of HSulf2 at HS NREs implicates the Sulfs as key regulators of this region of the chains, and concomitantly, the protein-binding events that occur there.
The cell surface glycoprotein γ-glutamyl transpeptidase (GGT) was isolated from healthy human kidney and liver to characterize
its glycosylation in normal human tissue in vivo. GGT is expressed by a single cell type in the kidney. The spectrum of N-glycans released from kidney GGT constituted a subset of the N-glycans identified from renal membrane glycoproteins. Recent advances in mass spectrometry enabled us to identify the microheterogeneity
and relative abundance of glycans on specific glycopeptides and revealed a broader spectrum of glycans than was observed among
glycans enzymatically released from isolated GGT. A total of 36 glycan compositions, with 40 unique structures, were identified
by site-specific glycan analysis. Up to 15 different glycans were observed at a single site, with site-specific variation
in glycan composition. N-Glycans released from liver membrane glycoproteins included many glycans also identified in the kidney. However, analysis
of hepatic GGT glycopeptides revealed 11 glycan compositions, with 12 unique structures, none of which were observed on kidney
GGT. No variation in glycosylation was observed among multiple kidney and liver donors. Two glycosylation sites on renal GGT
were modified exclusively by neutral glycans. In silico modeling of GGT predicts that these two glycans are located in clefts on the surface of the protein facing the cell membrane,
and their synthesis may be subject to steric constraints. This is the first analysis at the level of individual glycopeptides
of a human glycoprotein produced by two different tissues in vivo and provides novel insights into tissue-specific and site-specific glycosylation in normal human tissues.
Campylobacter jejuni is a gastrointestinal pathogen that is able to modify membrane and periplasmic proteins by the N-linked addition of a 7-residue glycan at the strict attachment motif (D/E)XNX(S/T). Strategies for a comprehensive analysis of the targets of glycosylation, however, are hampered by the resistance of the glycan-peptide bond to enzymatic digestion or β-elimination and have previously concentrated on soluble glycoproteins compatible with lectin affinity and gel-based approaches. We developed strategies for enriching C. jejuni HB93-13 glycopeptides using zwitterionic hydrophilic interaction chromatography and examined novel fragmentation, including collision-induced dissociation (CID) and higher energy collisional (C-trap) dissociation (HCD) as well as CID/electron transfer dissociation (ETD) mass spectrometry. CID/HCD enabled the identification of glycan structure and peptide backbone, allowing glycopeptide identification, whereas CID/ETD enabled the elucidation of glycosylation sites by maintaining the glycan-peptide linkage. A total of 130 glycopeptides, representing 75 glycosylation sites, were identified from LC-MS/MS using zwitterionic hydrophilic interaction chromatography coupled to CID/HCD and CID/ETD. CID/HCD provided the majority of the identifications (73 sites) compared with ETD (26 sites). We also examined soluble glycoproteins by soybean agglutinin affinity and two-dimensional electrophoresis and identified a further six glycosylation sites. This study more than doubles the number of confirmed N-linked glycosylation sites in C. jejuni and is the first to utilize HCD fragmentation for glycopeptide identification with intact glycan. We also show that hydrophobic integral membrane proteins are significant targets of glycosylation in this organism. Our data demonstrate that peptide-centric approaches coupled to novel mass spectrometric fragmentation techniques may be suitable for application to eukaryotic glycoproteins for simultaneous elucidation of glycan structures and peptide sequence.
We present a method to enrich for glycoproteins from proteomic samples. Sialylated glycoproteins were selectively periodate-oxidized, captured on hydrazide beads, trypsinized and released by acid hydrolysis of sialic acid glycosidic bonds. Mass spectrometric fragment analysis allowed identification of glycan structures, and additional fragmentation of deglycosylated ions yielded peptide sequence information, which allowed glycan attachment site and protein identification. We identified 36 N-linked and 44 O-linked glycosylation sites on glycoproteins from human cerebrospinal fluid.
Ovarian cancer causes more deaths than any other gynecological disorder. Perturbed glycosylation is one of the hallmarks of this malignancy. Kallikrein 6 (KLK6) elevation in serum is a diagnostic and prognostic indicator in ovarian cancer. The majority of ovarian carcinomas express high levels of KLK6, which diffuses into the circulation. Under physiological conditions, KLK6 is expressed highly in the central nervous system and found at high levels in cerebrospinal fluid from where it enters the circulation. Our aim was to characterize and compare the N-glycosylation status of this protein in ovarian cancer ascites fluid and cerebrospinal fluid. Anion-exchange chromatography was used to reveal different post-translational modifications on the two isoforms. Mobility gel shift Western blot analysis coupled with glycosidase digestion showed that the molecular weight difference between the two isoforms was because of differential glycosylation patterns. The presence of a single N-glycosylation site on KLK6 was confirmed by site-directed mutagenesis. Using a Sambucus nigra agglutinin-monoclonal antibody sandwich enzyme-linked immunosorbent assay approach, it was shown that ovarian cancer-derived KLK6 was modified with alpha2-6-linked sialic acid. The structure and composition of glycans of both KLK6 isoforms was elucidated by glycopeptide monitoring with electrospray ionization-Orbitrap tandem mass spectrometry. Therefore, the extensive and almost exclusive sialylation of KLK6 from ovarian cancer cells could lead to the development of an improved biomarker for the early diagnosis of ovarian carcinoma.
To examine the extent to which protein structure and tissue-type influence glycosylation, we have determined the oligosaccharide structures at each of the three glycosylation sites (Asn-23, 74 and 98) of the cell surface glycoprotein Thy-1 isolated from rat brain and thymus. The results show that there is tissue-specificity of glycosylation and that superimposed on this is a significant degree of site-specificity. On the basis of the site distribution of oligosaccharides, we find that no Thy-1 molecules are in common between the two tissues despite the amino acid sequences being identical. We suggest, therefore, that by controlling N-glycosylation a tissue creates an unique set of glycoforms (same polypeptide but with oligosaccharides that differ either in sequence or disposition). The structures at each of the three sites were also determined for the thymocyte Thy-1 that binds to lentil lectin (Thy-1 L+) and for that which does not (Thy-1 L-). Segregation of intact thymus Thy-1 into two distinct sets of glycoforms by lentil lectin was found to be due to the structures at site 74. Analysis of oligosaccharide structures at the 'passenger' sites (23 and 98) suggests that either Thy-1 L+ and Thy-1 L- molecules are made in different cell-types or that the biosynthesis of oligosaccharides at one site is influenced by the glycosylation at other sites.
Oligosaccharide patterns obtained from human IgG by hydrazinolysis were fairly constant from sample to sample. In contrast, a variety of oligosaccharide patterns were obtained from IgG myeloma proteins. Structural analysis of each oligosaccharide by sequential exoglycosidase digestion and methylation studied indicated that the different patterns of IgG myeloma proteins are produced by different degrees of incompleteness of the formation of a single sugar chain: Sia alpha 2 leads to Gal beta 1 leads to 4GlcNAc beta 1 leads to 2 Man alpha 1 leads to 6(Sia alpha 2 leads to Gal beta 1 leads to 4GlcNAc beta 1 leads to Man alpha 1 leads to 3)(GlcNAc beta 1 leads to 4)Man beta 1 leads to 4GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6)GlcNAcOT.
In Saccharomyces cerevisiae, transfer of N-linked oligosaccharides is immediately followed by trimming of ER-localized glycosidases. We analyzed the influence of specific oligosaccharide structures for degradation of misfolded carboxypeptidase Y (CPY). By studying the trimming reactions in vivo, we found that removal of the terminal alpha1,2 glucose and the first alpha1,3 glucose by glucosidase I and glucosidase II respectively, occurred rapidly, whereas mannose cleavage by mannosidase I was slow. Transport and maturation of correctly folded CPY was not dependent on oligosaccharide structure. However, degradation of misfolded CPY was dependent on specific trimming steps. Degradation of misfolded CPY with N-linked oligosaccharides containing glucose residues was less efficient compared with misfolded CPY bearing the correctly trimmed Man8GlcNAc2 oligosaccharide. Reduced rate of degradation was mainly observed for misfolded CPY bearing Man6GlcNAc2, Man7GlcNAc2 and Man9GlcNAc2 oligosaccharides, whereas Man8GlcNAc2 and, to a lesser extent, Man5GlcNAc2 oligosaccharides supported degradation. These results suggest a role for the Man8GlcNAc2 oligosaccharide in the degradation process. They may indicate the presence of a Man8GlcNAc2-binding lectin involved in targeting of misfolded glycoproteins to degradation in S. cerevisiae.
N-linked oligosaccharides arise when blocks of 14 sugars are added cotranslationally to newly synthesized polypeptides in
the endoplasmic reticulum (ER). These glycans are then subjected to extensive modification as the glycoproteins mature and
move through the ER via the Golgi complex to their final destinations inside and outside the cell. In the ER and in the early
secretory pathway, where the repertoire of oligosaccharide structures is still rather small, the glycans play a pivotal role
in protein folding, oligomerization, quality control, sorting, and transport. They are used as universal “tags” that allow
specific lectins and modifying enzymes to establish order among the diversity of maturing glycoproteins. In the Golgi complex,
the glycans acquire more complex structures and a new set of functions. The division of synthesis and processing between the
ER and the Golgi complex represents an evolutionary adaptation that allows efficient exploitation of the potential of oligosaccharides.
Lec13 cells, a variant Chinese hamster ovary cell line, were used to produce human IgG1 that were deficient in fucose attached
to the Asn297-linked carbohydrate but were otherwise similar to that found in IgG1 produced in normal Chinese hamster ovary cell lines
and from human serum. Lack of fucose on the IgG1 had no effect on binding to human FcγRI, C1q, or the neonatal Fc receptor.
Although no change in affinity was found for the His131 polymorphic form of human FcγRIIA, a slight improvement in binding was evident for FcγRIIB and the Arg131 FcγRIIA polymorphic form. In contrast, binding of the fucose-deficient IgG1 to human FcγRIIIA was improved up to 50-fold.
Antibody-dependent cellular cytotoxicity assays using purified peripheral blood monocytes or natural killer cells from several
donors showed enhanced cytotoxicity, especially evident at lower antibody concentrations. When combined with an IgG1 Fc protein
variant that exhibited enhanced antibody-dependent cellular cytotoxicity, the lack of fucose was synergistic.
Quantitative proteome profiling using stable isotope protein tagging and automated tandem mass spectrometry (MS/MS) is an emerging technology with great potential for the functional analysis of biological systems and for the detection of clinical diagnostic or prognostic marker proteins. Owing to the enormous complexity of proteomes, their comprehensive analysis is an as-yet-unresolved technical challenge. However, biologically or clinically important information can be obtained if specific, information-rich protein classes, or sub-proteomes, are isolated and analyzed. Glycosylation is the most common post-translational modification. Here we describe a method for the selective isolation, identification and quantification of peptides that contain N-linked carbohydrates. It is based on the conjugation of glycoproteins to a solid support using hydrazide chemistry, stable isotope labeling of glycopeptides and the specific release of formerly N-linked glycosylated peptides via peptide- N-glycosidase F (PNGase F). The recovered peptides are then identified and quantified by MS/MS. We applied the approach to the analysis of plasma membrane proteins and proteins contained in human blood serum.
Almost all of the key molecules involved in the innate and adaptive immune response are glycoproteins. In the cellular immune
system, specific glycoforms are involved in the folding, quality control, and assembly of peptide-loaded major histocompatibility
complex (MHC) antigens and the T cell receptor complex. Although some glycopeptide antigens are presented by the MHC, the
generation of peptide antigens from glycoproteins may require enzymatic removal of sugars before the protein can be cleaved.
Oligosaccharides attached to glycoproteins in the junction between T cells and antigen-presenting cells help to orient binding
faces, provide protease protection, and restrict nonspecific lateral protein-protein interactions. In the humoral immune system,
all of the immunoglobulins and most of the complement components are glycosylated. Although a major function for sugars is
to contribute to the stability of the proteins to which they are attached, specific glycoforms are involved in recognition
events. For example, in rheumatoid arthritis, an autoimmune disease, agalactosylated glycoforms of aggregated immunoglobulin
G may induce association with the mannose-binding lectin and contribute to the pathology.
The acquisition of high-resolution tandem mass spectra (MS/MS) is becoming more prevalent in proteomics, but most researchers employ peptide identification algorithms that were designed prior to this development. Here we demonstrate new software, Morpheus, designed specifically for high-mass accuracy data, based on a simple score that is little more than the number of matching products. For a diverse collection of datasets from a variety of organisms (E. coli, yeast, human) acquired on a variety of instruments (quadrupole-time of flight, ion trap-orbitrap, quadrupole-orbitrap) in different laboratories, Morpheus gives more spectrum, peptide, and protein identifications at a 1% false discovery rate (FDR) than Mascot, Open Mass Spectrometry Search Algorithm (OMSSA), and Sequest. Additionally, Morpheus is 1.5 to 4.6 times faster-depending on the dataset-than the next fastest algorithm, OMSSA. Morpheus was developed in C# .NET and is available free and open source under a permissive license.
N-Linked glycoproteins are involved in several diseases and are important as potential diagnostic molecules for biomarker discovery. Therefore, it is important to provide sensitive and reliable analytical methods to identify not only the glycoproteins but also the sites of glycosylation. Recently, numerous strategies to identify N-linked glycosylation sites have been described. These strategies have been applied to cell lines and several tissues with the aim of identifying many hundreds (or thousands) of glycosylation events. With high-throughput strategies however, there is always the potential for false positives. The confusion arises since the protein N-glycosidase F (PNGase F) reaction used to separate N-glycans from formerly glycosylated peptides catalyzes the cleavage and deamidates the asparagine residue. This is typically viewed as beneficial since it acts to highlight the modification site. We have evaluated this common large-scale N-linked glycoproteomic strategy and proved potential pitfalls using Escherichia coli as a model organism, since it lacks the N-glycosylation machinery found in mammalian systems and some pathogenic microbes. After isolation and proteolytic digestion of E. coli membrane proteins, we investigated the presence of deamidated asparagines. The results show the presence of deamidated asparagines especially with close proximity to a glycine residue or other small amino acid, as previously described for spontaneous in vivo deamidation. Moreover, we have identified deamidated peptides with incorporation of (18)O, showing the pitfalls of glycosylation site assignment based on deamidation of asparagine induced by PNGase F in (18)O-water in large-scale analyses. These data experimentally prove the need for more caution in assigning glycosylation sites and "new" N-linked consensus sites based on common N-linked glycoproteomics strategies without proper control experiments. Besides showing the spontaneous deamidation, we provide alternative methods for validation that should be used in such experiments.
Analysis of protein glycosylation is an important first step towards establishing the functions of glycans in health and disease. In contrast to N-glycans which are generally enzymatically released for analysis, there is no corresponding enzyme for O-glycan liberation. Therefore, O-glycans are generally released by chemical methods involving tedious procedures.
Here, a straightforward method for the combined release and labeling of O-linked glycans from glycoproteins is described. Dimethylamine serves as the releasing agent, and 1-phenyl-3-methyl-5-pyrazolone (PMP) is employed for a prompt reaction with the reducing end of the freshly released O-glycan structures via an aldol condensation followed by a Michael-type addition resulting in a 2:1 stoichiometry of PMP per glycan. Samples are analyzed by nanoLC coupled to mass spectrometry.
Mucin from bovine submaxillary gland was used as a model protein to evaluate and optimize the approach that was further applied to bile salt stimulated lipase (BSSL) isolated from human milk. Next to previously reported O-glycan structures two additional oligosaccharides could be detected for BSSL.
In conclusion, the facile protocol established is suitable for the analysis of complex O-linked oligosaccharides from various biological samples. This article is part of a Special Issue entitled Glycoproteomics.
Extracellular superoxide dismutase (EC-SOD), the major SOD isoenzyme in biological fluids, is known to be N-glycosylated and heterogeneous as was detected in most glycoproteins. However, only one N-glycan structure has been reported in recombinant human EC-SOD produced in Chinese hamster ovary (CHO) cells. Thus, a precise N-glycan profile of the recombinant EC-SOD is not available. In this study, we report profiling of the N-glycan in the recombinant mouse EC-SOD produced in CHO cells using high-resolution techniques, including the liberation of N-glycans by treatment with PNGase F, fluorescence labeling by pyridylamination, characterization by anion-exchange, normal and reversed phase-HPLC separation, and mass spectrometry. We succeeded in identifying 26 different types of N-glycans in the recombinant enzyme. The EC-SOD N-glycans were basically core-fucosylated (98.3% of the total N-glycan content), and were high mannose sugar chain, and mono-, bi-, tri-, and tetra-antennary complex sugar chains exhibiting varying degrees of sialylation. Four of the identified N-glycans were uniquely modified with a sulfate group, a Lewis(x) structure, or an α-Gal epitope. The findings will shed new light on the structure-function relationships of EC-SOD N-glycans.
The glycosylation profile of a recombinant protein is important because glycan moieties can play a significant role in the biological properties of the glycoprotein. Here we determined the site-specific N-glycosylation profile of human lactoferrin (hLF) and recombinant human lactoferrin (rhLF) expressed in the milk of transgenic cloned cattle. We used combined approaches of monosaccharide composition analysis, lectin blot, glycan permethylation and sequential exoglycosidase digestion and analyzed samples using high-performance ion chromatography and mass spectrometry (MS). N-glycans from hLF are comprised entirely of highly branched, highly sialylated and highly fucosylated complex-type structures, and many contain Lewis(x) epitopes. Six of these structures are reported here for the first time. However, N-glycans from rhLF are of the high mannose-, hybrid- and complex-type structures, with less N-acetylneuraminic acid and fucose. Some contain a terminal N-acetylgalactosamine-N-acetylglucosamine (LacdiNAc) disaccharide sequence. Monosaccharide composition analysis of rhLF revealed small amounts of N-glycolylneuraminic acid, which were not detected by MS. hLF and rhLF appear to be glycosylated at the same two sites: Asn138 and Asn479. The third putative glycosylation site, at Asn624, is unglycosylated in both hLF and rhLF. The relative abundance of each N-glycan at each site was also determined. The different N-glycosylation profile of rhLF when compared with that of hLF is in consistent with the widely held view that glycosylation is species- and tissue/cell-specific. These data provide an important foundation for further studies of glycan structure/function relationships for hLF and rhLF and help to better understand the glycosylation mechanism in bovine mammary epithelial cells.
Currently, scoring algorithms of many popular search engines for tandem mass spectrometry (MS/MS) data only partially utilize the information content of high mass accuracy MS/MS data. We have developed a new rescoring scheme, H-score, that employs high mass accuracy matching of all detected fragment ions to candidate peptide sequences in an abundance independent fashion. Peptides for which b or y ions are found for all or almost all backbone fragmentation sites are rewarded. For peptide hits generated by Mascot, rescoring proved to be particularly beneficial when applied on samples containing many different potential modifications. For a histone sample acquired on an Orbitrap Velos using HCD for peptide fragmentation, the H-score identified 24% more spectra at 0.01 false positive rate than Mascot scoring of spectra processed according to state-of-the-art methods and 61% better than Mascot scoring of unprocessed MS/MS spectra. For a low-abundance sample, where many weak spectra were detected, these numbers went up to 53 and 190%, respectively. When applied on a kinase-enriched sample containing only a few modifications, a smaller but still significant gain of 5% was observed.
Glycopeptide enrichment is a prerequisite to enable structural characterization of protein glycosylation in glycoproteomics. Here we present an improved method for glycopeptide enrichment based on zwitter-ionic hydrophilic interaction chromatography solid phase extraction (ZIC-HILIC SPE) in a microcolumn format. The method involves TFA ion pairing (IP) to increase the hydrophilicity difference between glycopeptides and nonglycosylated peptides. Three mobile phases were investigated, i.e., 2% formic acid (defined as IP(2% FA) ZIC-HILIC SPE), 0.1% TFA and 1% TFA (defined as IP(0.1% TFA) and IP(1% TFA) ZIC-HILIC SPE) all containing 80% acetonitrile. Samples of increasing complexities, i.e., digests of single glycoproteins, a five-glycoprotein mixture, and depleted plasma, were used in the study. The presence of TFA in the mobile phase significantly improved the glycopeptide enrichment for all complexities, as evaluated by enhanced glycopeptide detection using MALDI-TOF MS and RP-LC-ESI-MS/MS, e.g., the glycopeptide ion signals were increased by up to 3.7-fold compared to IP(2% FA) conditions. The enhanced glycopeptide detection was promoted by a substantial depletion of nonglycosylated peptides, offering an almost complete isolation of IgG glycopeptides using a single SPE enrichment step and a reduction from 711 nonglycosylated peptides observed in the IP(2% FA) ZIC-HILIC SPE retained plasma fraction, to only 157 and 97 when 0.1% and 1% TFA was used in the mobile phase. In conclusion, this systematic study has shown that TFA-containing mobile phases increase glycopeptide enrichment efficiency considerably for a broad range of sample complexities when using ZIC-HILIC SPE.
N-linked glycosylation is a biologically important protein modification, but only a small fraction of modification sites have been mapped. We developed a "filter aided sample preparation" (FASP)-based method in which glycopeptides are enriched by binding to lectins on the top of a filter and mapped 6367 N-glycosylation sites on 2352 proteins in four mouse tissues and blood plasma using high-accuracy mass spectrometry. We found 74% of known mouse N-glycosites and discovered an additional 5753 sites on a diverse range of proteins. Sites almost always have the N-!P-[S|T]-!P (where !P is not proline) and rarely the N-X-C motif or nonconsensus sequences. Combining the FASP approach with analysis of subcellular glycosite localization reveals that the sites always orient toward the extracellular space or toward the lumen of ER, Golgi, lysosome, or peroxisome. The N-glycoproteome contains a plethora of modification sites on factors important in development, organ-specific functions, and disease.
Despite the significance of glycoproteins for extracellular matrix assembly in cartilage tissue, little is known about the regulation of the chondrocyte glycophenotype under inflammatory conditions. The present study aimed to assess the effect of IL-1beta and TNF-alpha on specific features of the glycophenotype of primary human chondrocytes in vitro. Using LC-MS, we found that both cytokines increased overall sialylation of N- and O-glycans and induced a shift towards alpha-(2-->3)-linked sialic acid residues in chondrocyte glycoproteins. These results were supported by quantitative PCR showing increased expression of alpha-(2-->3) sialyltransferases in treated cells. Moreover, we found that both IL-1beta and TNF-alpha induced a considerable shift from oligomannosidic glycans towards complex-type N-glycans. In contrast, core alpha-(1-->6)-fucosylation of chondrocyte N-glycans was found to be reduced particularly by TNF-alpha. In summary, inflammatory conditions induce specific alterations of the chondrocyte glycophenotype which might affect cell-matrix interactions or the function of endogenous lectins.
Negative ion mode nanoliquid chromatography/mass spectrometry (nano-LC/MS) on porous graphitic carbon columns at pH 11 was studied and compared to capillary LC/MS at pH 8 for the analysis of neutral and acidic glycan alditols. Oligosaccharides were chromatographed with an acetonitrile gradient containing 0.04% ammonium hydroxide and analyzed with a linear ion trap mass spectrometer (LTQ) equipped with a modified nanospray interface. Analysis of acidic N- and O-glycan standards revealed that good quality MS/MS spectra could be obtained when loading 1-3 fmol, a 10-fold increase in sensitivity compared to capillary-LC/MS at pH 8. Analysis of a complex mixture of O-glycans from porcine colonic mucins with nano-LC/MS and MS/MS at high pH revealed 170 oligosaccharides in one analysis, predominantly corresponding to sulfated glycans with up to 11 residues. Analysis of the same sample with capillary-LC/MS showed a lower sensitivity for multiply sulfated glycans. Nano-LC/MS of O-linked oligosaccharides on MUC2 from a human colon biopsy also illustrated that the ionization of oligosaccharides with multiple sialic acid groups was increased compared to those with only one sialic acid residue. Nano-LC/MS at high pH is, thus, a highly sensitive approach for the analysis of acidic oligosaccharides.
We have previously reported that galectin-4, a tandem repeat-type galectin, regulates the raft-dependent delivery of glycoproteins to the apical brush border membrane of enterocyte-like HT-29 cells. N-Acetyllactos-amine-containing glycans, known as galectin ligands, were found enriched in detergent-resistant membranes. Here, we analyzed the potential contribution of N- and/or O-glycans in this mechanism. Structural studies were carried out on the brush border membrane-enriched fraction using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and nano-ESI-QTOF-MS/MS. The pattern of N-glycans was very heterogeneous, with the presence of high mannose- and hybrid-type glycans as well as a multitude of complex-type glycans. In contrast, the pattern of O-glycans was very simple with the presence of two major core type 1 O-glycans, sialylated and bisialylated T-antigen structures [Neu5Acalpha2-3Galbeta1-3GalNAc-ol and Neu5Acalpha2- 3Galbeta1-3(Neu5Acalpha2-6)GalNAc-ol]. Thus, N-glycans rather than O-glycans contain the N-acetyllactosamine recognition signals for the lipid raft-based galectin-4-dependent apical delivery. In the presence of 1-deoxymannojirimycin, a drug which inhibits the generation of hybrid-type or complex type N-glycans, the extensively O-glycosylated mucin-like MUC1 glycoprotein was not delivered to the apical brush border but accumulated inside the cells. Altogether, our data demonstrate the crucial role of complex N-glycans in the galectin-4-dependent delivery of glycoproteins to the apical brush border membrane of enterocytic HT-29 cells.
The structures of oligosaccharides of normal and pathological immunoglobulin G (IgG) are reported. Asparagine-linked neutral oligosaccharides were released by N-oligosaccharide glycopeptidase (almond) digestion. The reducing ends of the oligosaccharide chains thus obtained were aminated with a fluorescent reagent, 2-aminopyridine, and the mixture of pyridylamino derivatives of the oligosaccharides was separated by reverse-phase high-performance liquid chromatography. It was possible to separate 15 out of the 16 kinds of oligosaccharides that have been suggested to exist in normal human IgG. High-resolution proton nuclear magnetic resonance spectroscopy was used along with chemical methods to determine the structures of the separated oligosaccharides. It has been shown that in normal IgG a biantennary complex-type oligosaccharide with a fucose residue (formula; see text) is predominant and four kinds of oligosaccharides, which are biantennary with bisecting N-acetylglucosamine and without fucose residues, exist only in a very small quantity. The results obtained for normal IgG were compared with those obtained for three myeloma IgG proteins. It has been found that the most abundant species that exist in the pathological proteins analyzed in the present work lack one or two galactose residues at the nonreducing terminal. We show that the fractions of fucose-containing oligosaccharides are markedly decreased in the heavy-chain disease protein Per. It is of particular interest that in this paraprotein the major component is a biantennary complex-type oligosaccharide that lacks a fucose residue and an oligosaccharide with the structure (Formula: see text) exists as one of the most abundant components.
The adhesion domain of human CD2 bears a single N-linked carbohydrate. The solution structure of a fragment of CD2 containing the covalently bound high-mannose N-glycan [-(N-acetylglucosamine)2-(mannose)5-8] was solved by nuclear magnetic resonance. The stem and two of three branches of the carbohydrate structure are well defined and the mobility of proximal glycan residues is restricted. Mutagenesis of all residues in the vicinity of the glycan suggests that the glycan is not a component of the CD2-CD58 interface; rather, the carbohydrate stabilizes the protein fold by counterbalancing an unfavorable clustering of five positive charges centered about lysine-61 of CD2.
Protein structure and tissue type are known to influence glycosylation of proteins. We have previously investigated the N-glycans at each of the three glycosylation sites of the cell surface glycoprotein Thy-1 when isolated from rat brain and
thymocytes. Here we report a comparative analysis of the site-specific N-glycosylation patterns from rat (Asn 23, 74, 98), mouse (Asn 23, 75, 99) and human (Asn 23, 60, 100) neural Thy-1. Despite
considerable differences in amino acid sequence, the results show a remarkable conservation of the pattern of N-glycans at corresponding sites between the three species, as judged by chromatographic comparisons and glycosidase susceptibility.
This is particularly marked for sites at Asn 74/75 in rat/mouse and the equivalent site at 60 in human Thy-1, as well as for
sites at Asn 98/99 and 100, respectively. The sites at Asn 23 in rat/mouse also contained almost identical glycosylation paterns,
but at this site human Thy-1 showed significantly different glycosylation patterns. These site glycosylation patterns are
discussed in relation to the likely accessibility of the oligosaccharides for processing. It is known that within a species,
the glycosylation of Thy-1 is tissue specific; therefore, this degree of conservation of glycosylation of Thy-1 expressed
in the same tissue in different species is all the more striking, given the known variation between species in the amino acid
sequence of Thy-1. It is therefore proposed that neural cells have a particular requirement for specific surface carbohydrates
and that the Thy-1 polypep-tide serves as an appropriate carrier for these structures.
Glycosylation of proteins has been shown to play a role in a variety of cellular events. Thanks to recent advances in obtaining conformational constraints across glycosidic linkages, structural characterisation of glycoproteins has improved considerably. It is now becoming apparent that N-glycosylation of a folded protein can have a significant stabilising effect on large regions of the backbone structure.
Is there evidence that similar derangements in N-glycosylation contribute to autoimmunity in humans? There are rare carbohydrate deficiency syndromes in humans affecting proximal and largely essential steps in N-glycan synthesis, but these are associated with extremely severe, nonimmune multiorgan dysfunction early in life (Lowe and Freeze, 1999xNaturally occurring disorders of glycosylation. Lowe, J.B. and Freeze, H. See all References(Lowe and Freeze, 1999) and are not likely to be informative for autoimmune susceptibility. Humans with documented inactivating mutations in the MII or Hgat5 loci have not been reported, so their relevance to human autoimmune disease has not been studied. Linkage studies have not identified either as a clear candidate for an autoimmune susceptibility locus. Nonetheless, these loci, and their cognate N-glycans, represent important candidates for further study as contributors to the pathogenesis of systemic lupus erythematosis and other autoimmune syndromes.
Fringe modulates Notch signaling resulting in the establishment of compartmental boundaries in developing organisms. Fringe is a beta 3N-acetylglucosaminyltransferase (beta 3GlcNAcT) that transfers GlcNAc to O-fucose in epidermal growth factor-like repeats of Notch. Here we use five different Chinese hamster ovary cell glycosylation mutants to identify a key aspect of the mechanism of fringe action. Although the beta 3GlcNAcT activity of manic or lunatic fringe is shown to be necessary for inhibition of Jagged1-induced Notch signaling in a coculture assay, it is not sufficient. Fringe fails to inhibit Notch signaling if the disaccharide generated by fringe action, GlcNAc beta 3Fuc, is not elongated. The trisaccharide, Gal beta 4GlcNAc beta 3Fuc, is the minimal O-fucose glycan to support fringe modulation of Notch signaling. Of six beta 4galactosyltransferases (beta 4GalT) in Chinese hamster ovary cells, only beta 4GalT-1 is required to add Gal to GlcNAc beta 3Fuc, identifying beta 4GalT-1 as a new modulator of Notch signaling.
The functional consequences of glycan structural changes associated with cellular differentiation are ill defined. Herein, we investigate the role of glycan adducts to the O-glycosylated polypeptide stalk tethering the CD8alphabeta coreceptor to the thymocyte surface. We show that immature CD4(+)CD8(+) double-positive thymocytes bind MHCI tetramers more avidly than mature CD8 single-positive thymocytes, and that this differential binding is governed by developmentally programmed O-glycan modification controlled by the ST3Gal-I sialyltransferase. ST3Gal-I induction and attendant core 1 sialic acid addition to CD8beta on mature thymocytes decreases CD8alphabeta-MHCI avidity by altering CD8alphabeta domain-domain association and/or orientation. Hence, glycans on the CD8beta stalk appear to modulate the ability of the distal binding surface of the dimeric CD8 globular head domains to clamp MHCI.
An anti-human interleukin 5 receptor (hIL-5R) humanized immunoglobulin G1 (IgG1) and an anti-CD20 chimeric IgG1 produced by rat hybridoma YB2/0 cell lines showed more than 50-fold higher antibody-dependent cellular cytotoxicity (ADCC) using purified human peripheral blood mononuclear cells as effector than those produced by Chinese hamster ovary (CHO) cell lines. Monosaccharide composition and oligosaccharide profiling analysis showed that low fucose (Fuc) content of complex-type oligosaccharides was characteristic in YB2/0-produced IgG1s compared with high Fuc content of CHO-produced IgG1s. YB2/0-produced anti-hIL-5R IgG1 was subjected to Lens culinaris aggulutin affinity column and fractionated based on the contents of Fuc. The lower Fuc IgG1 had higher ADCC than the IgG1 before separation. In contrast, the content of bisecting GlcNAc of the IgG1 affected ADCC much less than that of Fuc. In addition, the correlation between Gal and ADCC was not observed. When the combined effect of Fuc and bisecting GlcNAc was examined in anti-CD20 IgG1, only a severalfold increase of ADCC was observed by the addition of GlcNAc to highly fucosylated IgG1. Quantitative PCR analysis indicated that YB2/0 cells had lower expression level of FUT8 mRNA, which codes alpha1,6-fucosyltransferase, than CHO cells. Overexpression of FUT8 mRNA in YB2/0 cells led to an increase of fucosylated oligosaccharides and decrease of ADCC of the IgG1. These results indicate that the lack of fucosylation of IgG1 has the most critical role in enhancement of ADCC, although several reports have suggested the importance of Gal or bisecting GlcNAc and provide important information to produce the effective therapeutic antibody.
We describe here a strategy for the large-scale identification of N-glycosylated proteins from a complex biological sample. The approach, termed isotope-coded glycosylation-site-specific tagging (IGOT), is based on the lectin column-mediated affinity capture of a set of glycopeptides generated by tryptic digestion of protein mixtures, followed by peptide-N-glycosidase-mediated incorporation of a stable isotope tag, 18O, specifically into the N-glycosylation site. The 18O-tagged peptides are then identified by multi-dimensional liquid chromatography-mass spectrometry (LC-MS)-based technology. The application of this method to the characterization of N-linked high-mannose and/or hybrid-type glycoproteins from an extract of Caenorhabditis elegans proteins allowed the identification of 250 glycoproteins, including 83 putative transmembrane proteins, with the simultaneous determination of 400 unique N-glycosylation sites. Because the method is applicable to the systematic identification of a wide range of glycoproteins, it should facilitate basic glycobiology research and may be useful for diagnostic applications, such as genome-wide screening for disease-related glycoproteins.
material. The studywas initiated partlyinordertoinvestigate thenature oftheprotein-carbohydrate linkage, butthisgoal couldnot be achievedby themethodsthen available. Someprogress hasbeenmade inthis problemby more recentstudies and Johansen, Marshall & Neuberger (1960) havegiventhemost probable values forthemannose,glucosamine and acetyl contents ofthewholeprotein andofaglycopeptide isolated fromit.Theprobability thatthese aretheonlysugarspresentwasindicated. Itis thepurposeofthispapertodescribe thepreparationand some oftheproperties ofthisglycopeptide, andtoconsider thenatureofthechemical bondlinking thecarbohydrate to theprotein. A briefdescription ofthisworkwas reported earlier (Johansen, Marshall & Neuberger, 1958). Cunningham, Nuenke& Nuenke (1957)and Jevons(1958) havealsogivenshortaccountsof their findings onthesamesubject.
An approach for the characterization of glycosylation sites and oligosaccharide heterogeneity in glycoproteins based on a combination of nonspecific proteolysis, deglycosylation, and matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FT MS) is described. Glycoproteins were digested with Pronase yielding primarily glycopeptides and amino acids. Nonglycosylated peptide fragments were susceptible to complete Pronase digestion to their constituent amino acids. Steric hindrance prohibited the digestion of the peptide moiety attached to the glycan. Glycopeptides were desalted and concentrated using solid-phase extraction and analyzed by MALDI MS. The oligosaccharides were also analyzed by MALDI MS after releasing the glycans from glycoproteins using PNGase F. The peptide moiety of the glycopeptides was identified by subtracting the masses of the glycans derived from PNGase F treatment from the masses of the glycopeptides. The experimental strategy was validated using glycoproteins with known oligosaccharide structures, ribonuclease B and chicken ovalbumin. This procedure was then used to determine the N-glycosylation sites and site heterogeneity of a glycoprotein whose glycosylation pattern was unknown, namely, the Xenopus laevis egg cortical granule lectin. This procedure is useful for determining protein site heterogeneity and structural heterogeneities of the oligosaccharide moiety of glycoproteins.
Characterization of glycoproteins using mass spectrometry ranges from determination of carbohydrate-protein linkages to the full characterization of all glycan structures attached to each glycosylation site. In a novel approach to identify N-glycosylation sites in complex biological samples, we performed an enrichment of glycosylated peptides through hydrophilic interaction liquid chromatography (HILIC) followed by partial deglycosylation using a combination of endo-beta-N-acetylglucosaminidases (EC 3.2.1.96). After hydrolysis with these enzymes, a single N-acetylglucosamine (GlcNAc) residue remains linked to the asparagine residue. The removal of the major part of the glycan simplifies the MS/MS fragment ion spectra of glycopeptides, while the remaining GlcNAc residue enables unambiguous assignment of the glycosylation site together with the amino acid sequence. We first tested our approach on a mixture of known glycoproteins, and subsequently the method was applied to samples of human plasma obtained by lectin chromatography followed by 1D gel-electrophoresis for determination of 62 glycosylation sites in 37 glycoproteins.