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Assessment of lectin and HILIC based enrichment protocols for characterization of serum glycoproteins by mass spectrometry
Abstract
Protein glycosylation is a common post-translational modification that is involved in many biological processes, including cell adhesion, protein-protein and receptor-ligand interactions. The glycoproteome constitutes a source for identification of disease biomarkers since altered protein glycosylation profiles are associated with certain human ailments. Glycoprotein analysis by mass spectrometry of biological samples, such as blood serum, is hampered by sample complexity and the low concentration of the potentially informative glycopeptides and -proteins. We assessed the utility of lectin-based and HILIC-based affinity enrichment techniques, alone or in combination, for preparation of glycoproteins and glycopeptides for subsequent analysis by MALDI and ESI mass spectrometry. The methods were successfully applied to human serum samples and a total of 86 N-glycosylation sites in 45 proteins were identified using a mixture of three immobilized lectins for consecutive glycoprotein enrichment and glycopeptide enrichment. The combination of lectin affinity enrichment of glycoproteins and subsequent HILIC enrichment of tryptic glycopeptides identified 81 N-glycosylation sites in 44 proteins. A total of 63 glycosylation sites in 38 proteins were identified by both methods, demonstrating distinct differences and complementarity. Serial application of custom-made microcolumns of mixed, immobilized lectins proved efficient for recovery and analysis of glycopeptides from serum samples of breast cancer patients and healthy individuals to assess glycosylation site frequencies.
... Similar to other PTMs, specific enrichment is essential to capture the often low abundance glycopeptides. Several enrichment methods, including lectin affinity [13,14], hydrophilic interaction [15] and solid phase extraction using hydrazide chemistry [16], have been developed and applied for characterizing N-glycoproteins and N-glycosites [17]. ...
... The two cell lines are derived from the same genetic background [18]. For improved coverage, hydrazide chemistry [19] and zic-HILIC [15] were tested for the enrichment of N-glycopeptides. After duplicate biological analyses, a total of 1,213 unique N-glycosites from 611 N-glycoproteins from both cell lines were confidently identified. ...
... The digested peptides were enriched with zic-HILIC media by the procedure reported by Calvano [15] with slight modifications. Briefly, first, the C8 disk was inserted into a 200 μL tip. ...
Cancer cell metastasis is a major cause of cancer death. Unfortunately, the underlying molecular mechanisms remain unknown, which results in the lack of efficient diagnosis, therapy and prevention approaches. Nevertheless, the dysregulation of the cancer cell secretome is known to play key roles in tumor transformation and progression. The majority of proteins in the secretome are secretory proteins and membrane-released proteins, and, mostly, the glycosylated proteins. Until recently, few studies have explored protein N-glycosylation changes in the secretome, although protein glycosylation has received increasing attention in the study of tumor development processes. Here, the N-glycoproteins in the secretome of two human hepatocellular carcinoma (HCC) cell lines with low (MHCC97L) or high (HCCLM3) metastatic potential were investigated with a in-depth characterization of the N-glycosites by combining two general glycopeptide enrichment approaches, hydrazide chemistry and zwitterionic hydrophilic interaction chromatography (zic-HILIC), with mass spectrometry analysis. A total of 1,213 unique N-glycosites from 611 N-glycoproteins were confidently identified. These N-glycoproteins were primarily localized to the extracellular space and plasma membrane, supporting the important role of N-glycosylation in the secretory pathway. Coupling label-free quantification with a hierarchical clustering strategy, we determined the differential regulation of several N-glycoproteins that are related to metastasis, among which AFP, DKK1, FN1, CD151 and TGFβ2 were up-regulated in HCCLM3 cells. The inclusion of the well-known metastasis-related proteins AFP and DKK1 in this list provides solid supports for our study. Further western blotting experiments detecting FN1 and FAT1 confirmed our discovery. The glycoproteome strategy in this study provides an effective means to explore potential cancer biomarkers.
... Another type of MLAC containing ConA, WGA and Sambucus nigra agglutinin (SNA) resulted in the identification of 45 different proteins in the serum of sick patients only [130]. ConA and WGA were applied in another study to assess BCa progression in which the glycoproteomic profile of premalignant and malignant cells with metastatic potential was evaluated. ...
Poor lifestyle choices and genetic predisposition are factors that increase the number of cancer cases, one example being breast cancer, the third most diagnosed type of malignancy. Currently, there is a demand for the development of new strategies to ensure early detection and treatment options that could contribute to the complete remission of breast tumors, which could lead to increased overall survival rates. In this context, the glycans observed at the surface of cancer cells are presented as efficient tumor cell markers. These carbohydrate structures can be recognized by lectins which can act as decoders of the glycocode. The application of plant lectins as tools for diagnosis/treatment of breast cancer encompasses the detection and sorting of glycans found in healthy and malignant cells. Here, we present an overview of the most recent studies in this field, demonstrating the potential of lectins as: mapping agents to detect differentially expressed glycans in breast cancer, as histochemistry/cytochemistry analysis agents, in lectin arrays, immobilized in chromatographic matrices, in drug delivery, and as biosensing agents. In addition, we describe lectins that present antiproliferative effects by themselves and/or in conjunction with other drugs in a synergistic effect.
... Glycoproteins or glycopeptides can be enriched by methods based on chemical and biological interactions, in which stationary phases containing charged or hydrophilic groups specific to glycans in their structures [ 40 , 41 ]. Hydrophilic interaction liquid chromatography (HILIC), lectin affinity, immobilized metal/metal oxide affinity, boronate affinity, and hydrazide chemistry are widely used methods for the selective enrichment of glycoproteins or glycopeptides [42][43][44][45][46][47][48] . ...
Aberrant glycosylation has been associated with the human disease since it involves crucial mechanisms such as tumor onset, development, and progression. This review focuses on the most commonly used sample preparation methods for standard glycoprotein, serum, cell, and tissue samples in N-glycan analysis studies. We aim to provide readers with an overview of N-glycomics sample preparation methods for profiling N-glycans. From this perspective, this review illustrates the main strategies of N-glycan analysis using different techniques. The molecular information obtained from glycomics analysis has allowed new visions into the mechanism concerning aberrant glycosylation.
... F o r e x a m p l e , immunodepletion technologies and ProteoMiner protein enrichment methods have been used to remove abundant plasma proteins (5)(6)(7). Many enrichment materials have been developed to remove nonglycopeptides in plasma (4,8). These methods and materials are beneficial to the study of serum glycoproteomics. ...
Monoclonal immunoglobulin produced by clonal plasma cells is the main cause in multiple myeloma and monoclonal gammopathy of renal significance. Because of the complicated purification method and the low stoichiometry of purified protein and glycans, site-specific N-glycosylation characterization for monoclonal immunoglobulin is still challenging. To profile the site-specific N-glycosylation of monoclonal immunoglobulins is of great interest. Therefore, in this study, we presented an integrated workflow for micro monoclonal IgA and IgG purification from patients with multiple myeloma in the HYDRASYS system, in-agarose-gel digestion, LC-MS/MS analysis without intact N-glycopeptide enrichment, and compared the identification performance of different mass spectrometry dissociation methods (EThcD-sceHCD, sceHCD, EThcD and sceHCD-pd-ETD). The results showed that EThcD-sceHCD was a better choice for site-specific N-glycosylation characterization of micro in-agarose-gel immunoglobulins (~2 μg) because it can cover more unique intact N-glycopeptides (37 and 50 intact N-glycopeptides from IgA1 and IgG2, respectively) and provide more high-quality spectra than sceHCD, EThcD and sceHCD-pd-ETD. We demonstrated the benefits of the alternative strategy in site-specific N-glycosylation characterizing micro monoclonal immunoglobulins obtained from bands separated by electrophoresis. This work could promote the development of clinical N-glycoproteomics and related immunology.
... After the non-glycosylated proteins have been washed through, the glycosylated proteins are released using an acidic solution [115]. HILIC, as described above, has also been applied for the enrichment of glycopeptides [118]. ...
Simple Summary
This review discusses current techniques used to study post-translational protein modifications (PTMs) and their contribution to blood cancer research. Blood cancer is an umbrella term for cancers that affect blood cells and organs of the hematopoietic system, accounting for approximately 10% of all cancer diagnoses. PTMs are crucial for the normal functioning of cells via the regulation of protein structure, function, and localization. Mass spectrometry and antibody-based techniques are powerful analytical tools used for the detection and quantitation of PTMs such as phosphorylation. The application of these techniques in blood cancer research facilitates the identification of abnormal PTMs that contribute to cancer development and progression by promoting cancer cell growth, survival, and invasion. Many FDA-approved blood cancer treatments exert their anti-cancer effects by targeting protein modifications, thus emphasizing the importance of PTM-focused research in the identification of novel biomarkers and therapeutic targets to improve blood cancer survival outcomes.
Abstract
Post-translational modifications (PTMs) add a layer of complexity to the proteome through the addition of biochemical moieties to specific residues of proteins, altering their structure, function and/or localization. Mass spectrometry (MS)-based techniques are at the forefront of PTM analysis due to their ability to detect large numbers of modified proteins with a high level of sensitivity and specificity. The low stoichiometry of modified peptides means fractionation and enrichment techniques are often performed prior to MS to improve detection yields. Immuno-based techniques remain popular, with improvements in the quality of commercially available modification-specific antibodies facilitating the detection of modified proteins with high affinity. PTM-focused studies on blood cancers have provided information on altered cellular processes, including cell signaling, apoptosis and transcriptional regulation, that contribute to the malignant phenotype. Furthermore, the mechanism of action of many blood cancer therapies, such as kinase inhibitors, involves inhibiting or modulating protein modifications. Continued optimization of protocols and techniques for PTM analysis in blood cancer will undoubtedly lead to novel insights into mechanisms of malignant transformation, proliferation, and survival, in addition to the identification of novel biomarkers and therapeutic targets. This review discusses techniques used for PTM analysis and their applications in blood cancer research.
... It offers several advantages such as shorter sample preparation time, ultrafast analysis due to low column backpressure and improved MS sensitivity. HILIC with online ESI-MS has been used widely for the analysis of released glycans (Luo et al. 2009;Mauko et al. 2011;Ruhaak et al. 2008;Zauner et al. 2011), glycopeptides (Calvano et al. 2008;Wohlgemuth et al. 2009;Zauner et al. 2010), GAG oligosaccharides (Huang et al. 2011;Kailemia et al. 2014;Staples et al. 2010). For GAG disaccharide analysis the challenge with HILIC is to find mobile phase conditions that achieve efficient retention of disaccharides containing a range of 0-4 sulfate groups. ...
The ECM is a complex molecular network that surrounds all cells and consists of proteins, glycoproteins, hyaluronan, glycosaminoglycans (GAGs), and proteoglycans (PGs). GAGs and PGs play vital roles in ECM-related processes such as cell migration, proliferation, adhesion, and differentiation. Among the omics technologies, including genomics, transcriptomics, and proteomics, glycomics is the least mature. Over the past two decades, with efforts from glycoscientists around world, and the advent of new glyco-techonologies, databases, tools, and methods, much progress has been made. Now, the focus is to integrate proteomics and glycomics domains in a new platform to analyze and characterize biomolecule classes and define their structural and functional roles. Towards this end, we have developed approaches that integrate analysis of GAGs and proteins towards an end goal of elucidating pathophysiological mechanisms to inform development of disease therapies and regenerative medicine. In this chapter, we provide a historical overview of our groups’ methods for glycomics, glycoproteomics, and proteomics of key ECM constituents, i.e., GAGs and PGs, as reported over the past decade.
... One aliquot (to the right) is treated with releasing glycosidase or glycosylamidase and the glycans and deglycosylated peptides are collected separately. The second aliquot, containing intact glycopeptides (left), can be subjected to glycopeptide enrichment via HILIC, porous graphitized carbon, lectin affinity, size exclusion chromatography or other selective process (Calvano, Zambonin, & Jensen, 2008;Wohlgemuth et al., 2010;Wang, Wu, & Khoo, 2011;. This step, while not required, can be used to decrease content of the more highly ionizable peptides and other contaminating compounds allowing for improved detection of the hydrophilic and relatively poorly ionizable glycopeptides . ...
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.
... Despite the technical improvements to MS instruments, it is still challenging to analyze phosphorylated or glycosylated peptides by direct LC-MS without reducing sample complexity, due to the sub-stoichiometric level abundance of the two PTMs and the poor ionization efficiency in positive ion mode MS derived from additional negative charges of these PTMs. Therefore, numerous enrichment techniques have been developed targeting phosphorylated or glycosylated peptides, including metal oxide affinity chromatography (MOAC) such as TiO 2 and ZrO 2 enrichment [36,37], immobilized metal affinity chromatography (IMAC) [38][39][40][41][42], lectin affinity chromatography-based enrichment [43,44], hydrazide chemistry-based SPE method [45][46][47][48], boronic acid enrichment method [49][50][51], and hydrophilic interaction chromatography (HILIC) [52][53][54][55]. Most of the methods listed above were designed to target phosphorylation or glycosylation alone and each has its own limitations. ...
Simultaneous enrichment of glyco- and phosphopeptides will benefit the studies of biological processes regulated by these posttranslational modifications (PTMs). It will also reveal potential crosstalk between these two ubiquitous PTMs. Unlike custom-designed multifunctional solid phase extraction (SPE) materials, operating strong anion exchange (SAX) resin in electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) mode provides a readily available strategy to analytical labs for enrichment of these PTMs for subsequent mass spectrometry (MS)-based characterization. However, the choice of mobile phase has largely relied on empirical rules from hydrophilic interaction chromatography (HILIC) or ion-exchange chromatography (IEX) without further optimization and adjustments. In this study, ten mobile phase compositions of ERLIC were systematically compared; the impact of multiple factors including organic phase proportion, ion pairing reagent, pH, and salt on the retention of glycosylated and phosphorylated peptides was evaluated. This study demonstrated good enrichment of glyco- and phosphopeptides from the nonmodified peptides in a complex tryptic digest. Moreover, the enriched glyco- and phosphopeptides elute in different fractions by orthogonal retention mechanisms of hydrophilic interaction and electrostatic interaction in ERLIC, maximizing the LC-MS identification of each PTM. The optimized mobile phase can be adapted to the ERLIC HPLC system, where the high resolution in separating multiple PTMs will benefit large-scale MS-based PTM profiling and in-depth characterization.
... Hydrophilic interaction liquid chromatography (HILIC) is a separation technique that has been extensively employed to separate/enrich glycopeptides (Calvano et al., 2008;Lam et al., 2010;Segu et al., 2010;Parker et al., 2013;Dedvisitsakul et al., 2014;Khatri et al., 2014;Qin et al., 2017;Lin et al., 2018). Contrary to reverse-phase chromatography, the stationary phase of HILIC is very hydrophilic, allowing binding of hydrophilic analytes to the column. ...
Protein glycosylation is ubiquitous in biological systems and plays essential roles in many cellular events. Global and site‐specific analysis of glycoproteins in complex biological samples can advance our understanding of glycoprotein functions and cellular activities. However, it is extraordinarily challenging because of the low abundance of many glycoproteins and the heterogeneity of glycan structures. The emergence of mass spectrometry (MS)‐based proteomics has provided us an excellent opportunity to comprehensively study proteins and their modifications, including glycosylation. In this review, we first summarize major methods for glycopeptide/glycoprotein enrichment, followed by the chemical and enzymatic methods to generate a mass tag for glycosylation site identification. We next discuss the systematic and quantitative analysis of glycoprotein dynamics. Reversible protein glycosylation is dynamic, and systematic study of glycoprotein dynamics helps us gain insight into glycoprotein functions. The last part of this review focuses on the applications of MS‐based proteomics to study glycoproteins in different biological systems, including yeasts, plants, mice, human cells, and clinical samples. Intact glycopeptide analysis is also included in this section. Because of the importance of glycoproteins in complex biological systems, the field of glycoproteomics will continue to grow in the next decade. Innovative and effective MS‐based methods will exponentially advance glycoscience, and enable us to identify glycoproteins as effective biomarkers for disease detection and drug targets for disease treatment. © 2019 Wiley Periodicals, Inc. Mass Spec Rev 9999: XX–XX, 2019.
... Many studies have compared the efficiency and specificity of different glycopeptide enrichment methods (Calvano, Zambonin, & Jensen, 2008;Ishihara, Fukuda, Morita, Takinami, Okamoto, Nishimura et al., 2011;Lewandrowski, Moebius, Walter, & Sickmann, 2006;Song et al., 2014;Wohlgemuth, Karas, Eichhorn, Hendriks, & Andrecht, 2009; L. G. C. Zhang, Ye, Xue, Shu, Zhou, Ji et al., 2016;H. Zhang, Guo, Li, Datta, Park, Yang et al., 2010;Y. ...
Protein glycosylation is one of the most important posttranslational modifications. Numerous biological functions are related to protein glycosylation. However, analytical challenges remain in the glycoprotein analysis. To overcome the challenges associated with glycoprotein analysis, many analytical techniques were developed in recent years. Enrichment methods were used to improve the sensitivity of detection, while HPLC and mass spectrometry methods were developed to facilitate the separation of glycopeptides/proteins and enhance detection, respectively. Fragmentation techniques applied in modern mass spectrometers allow the structural interpretation of glycopeptides/proteins, while automated software tools started replacing manual processing to improve the reliability and throughput of the analysis. In this chapter, the current methodologies of glycoprotein analysis were discussed. Multiple analytical techniques are compared, and advantages and disadvantages of each technique are highlighted.
... Unlike N-glycosylation, there is no robust means of removing the glycan prior to mass spectrometric analysis, and determinations of O-linked glycopeptides and proteins can be quite challenging [64]. Enrichment of glycopeptides and glycoproteins is typically accomplished using lectin-affinity chromatography [65][66][67][68]. The analysis of carbohydrate heterogeneity for a specific glycopeptide site is best performed in a stepwise manner by treating the proteolytic sample with appropriate enzymes [69]. ...
... The digested peptides were enriched with zic-HILIC media following the procedure reported by Calvano [64] with slight modifications. Briefly, first, the C8 disk was inserted into a 200-μL tip. ...
Cancer cell metastasis is a major cause of cancer fatality. But the underlying molecular mechanisms remain incompletely understood, which results in the lack of efficient diagnosis, therapy and prevention approaches. Here, we report a systematic study on the secretory proteins (secretome) and secretory N-glycoproteins (N-glycosecretome) of four human hepatocellular carcinoma (HCC) cell lines with different metastatic potential, to explore the molecular mechanism of metastasis and supply the clues for effective measurement of diagnosis and therapy. Totally, 6242 unique gene products (GPs) and 1637 unique N-glycosites from 635 GPs were confidently identified. About 4000 GPs on average were quantified in each of the cell lines, 1156 of which show differential expression (p<0.05). Ninety-nine percentage of the significantly altered proteins were secretory proteins and proteins correlated to cell movement were significantly activated with the increasing of metastatic potential of the cell lines. Twenty-three GPs increased both in the secretome and the N-glycosecretome were chosen as candidates and verified by western blot analysis, and 10 of them were chosen for immunohistochemistry (IHC) analysis. The cumulative survival rates of the patients with candidate (FAT1, DKK3) suggested that these proteins might be used as biomarkers for HCC diagnosis. In addition, a comparative analysis with the published core human plasma database (1754 GPs) revealed that there were 182 proteins not presented in the human plasma database but identified by our studies, some of which were selected and verified successfully by western blotting in human plasma.
... Binding of acidic peptides to TiO2 can also be reduced by sample binding in the presence of 2,5- dihydroxybenzoic acid (DHB) or glutamate (Wu et al., 2007). Hydrophilic interaction chromatography (HILIC) is usually used for the semi-selective enrichment of glycopeptides (Calvano et al., 2008; Picariello et al., 2008). Enrichment of glycans has been demonstrated to occur via a number of mechanisms including hydrogen bonding and ionic interactions. ...
Addition or removal of a functional group from an amino acid side chain, the modification of protein termini, cleavage of the synthesized polypeptide chain or the covalent cross-linking between separate protein domains is known as post-translational modification (PTM). Several proteins, particularly those derived from eukaryotic sources, undergo covalent modification either during their ribosomal synthesis or (more usually) after synthesis is complete, this is known as co-translational and post-translational modification. The stage during protein synthesis/ maturation at which the co- or post-translational modification occurs evidently differs but both are often referred as post-translational modifications (PTMs). These modifications invariably influence some structural aspect or functional role of the affected protein (Walsh 2009). Posttranslational
modification provides a dynamic mechanism for regulation of protein function. These well expand the chemical diversity of functional groups as well as regulatory potential ahead of those defined by the standard 20 amino acid side chains. Many cellular proteins undergo considerable amounts of Post-translational Modifications (PTMs) in response to certain stimuli and this dynamic process occurs in various cell compartments to dictate the fate and activity of the modified proteins.
... 16 Glycoprotein/glycopeptide enrichments using lectin or hydrophilic interaction LC (HILIC) are also common. 44,50 However, there are several concerns regarding these enrichment procedures. The most prominent ones include sample loss, reproducibility, and potentially enrichment bias for particular types of glycans. ...
A comprehensive glycan map was constructed for the top eight abundant glycoproteins in plasma using both specific and non-specific enzyme digestions followed by nano LC-Chip/QTOF mass spectrometry (MS) analysis. Glycopeptides were identified using an in-house software tool, GPFinder. A sensitive and reproducible multiple reaction monitoring (MRM) technique on a triple quadrupole MS was developed and applied to quantify immunoglobulins G, A, M, and their site-specific glycans simultaneously and directly from human serum/plasma without protein enrichments. A total of 64 glycopeptides and 15 peptides were monitored for IgG, IgA, and IgM in a 20-min UPLC gradient. The absolute protein contents were quantified using peptide calibration curves. The glycopeptide ion abundances were normalized to the respective protein abundances to separate protein glycosylation from protein expression. This technique yields higher method reproducibility and less sample loss when compared with the quantitation method that involves protein enrichments. The absolute protein quantitation has a wide linear range (3-4 orders of magnitude) and low limit of quantitation (femtomole level). This rapid and robust quantitation technique, which provides quantitative information for both proteins and glycosylation, will further facilitate disease biomarker discoveries.
... Therefore, it can be expected that a method for the systematic and quantitative analysis of cell surface sialoglycoproteins would be very useful for the detection of new potential diagnostic markers and therapeutic targets for malignant gliomas [20]. To detect low abundant glycosylated surface membrane proteins or peptides in complex mixtures, specific enrichment methods have to be applied, most commonly based on lectin affinity chromatography212223 , hydrophilic interaction chroma- tography [24,25], titanium dioxide chromatography [26,27] or chemical linkage of the sugar moiety to surfaces282930. Recently, a technology termed ''bioorthogonal chemical reporter'' (BOCR) strategy has been developed by Bertozzi et al. ...
Glycosylation is an incredibly common and diverse post‐translational modification that contributes widely to cellular health and disease. Mass spectrometry is the premier technique to study glycoproteins; however, glycoproteomics has lagged behind traditional proteomics due to the challenges associated with studying glycosylation. For instance, glycans dissociate by collision‐based fragmentation, thus necessitating electron‐based fragmentation for site‐localization. The vast glycan heterogeneity leads to lower overall abundance of each glycopeptide, and often, ion suppression is observed. One of the biggest issues facing glycoproteomics is the lack of reliable software for analysis, which necessitates manual validation and serves as a massive bottleneck in data processing. Here, I will discuss each of these challenges and some ways in which the field is attempting to address them, along with perspectives on how I believe we should move forward.
Sugars play important roles in numerous biological processes, from providing energy to modifying proteins to alter their function. Glycosylation, the attachment of a sugar residue to a protein, is the most common post translational modification. Identifying the glycans on a protein is a useful tool both for pharmaceutical development as well as probing the proteome and glycome further. Sugars, however, are difficult analytes to probe due to their isomeric nature. In this work, Raman spectroscopy and surface enhanced Raman spectroscopy (SERS) are used to identify different monosaccharide species based on the vibrational modes of these isomeric analytes. The weak scattering of the sugars was overcome through conjugation with phenylboronic acid to provide a larger Raman scattering cross section and induce slight changes in the observed spectra associated with the structure of the monosaccharides. Spontaneous Raman, SERS in flow, and static SERS detection were performed in order to discriminate between arabinose, fructose, galactose, glucose, mannose, and ribose, as well as provide a method for identification and quantification for these sugar conjugates.
Post-translational modifications (PTMs) regulate complex biological processes through the modulation of protein activity, stability, and localization. Insights into the specific modification type and localization within a protein sequence can help ascertain functional significance. Computational models are increasingly demonstrated to offer a low-cost, high-throughput method for comprehensive PTM predictions. Algorithms are optimized using existing experimental PTM data, thus accurate prediction performance relies on the creation of robust datasets. Herein, advancements in mass spectrometry-based proteomics technologies to maximize PTM coverage are reviewed. Further, requisite experimental validation approaches for PTM predictions are explored to ensure that follow-up mechanistic studies are focused on accurate modification sites.
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
Protein glycosylation fundamentally impacts biological processes. Nontemplated biosynthesis introduces unparalleled complexity into glycans that needs tools to understand their roles in physiology. The era of quantitative biology is a great opportunity to unravel these roles, especially by mass spectrometry glycoproteomics. However, with high sensitivity come stringent requirements on tool specificity. Bioorthogonal metabolic labeling reagents have been fundamental to studying the cell surface glycoproteome but typically enter a range of different glycans and are thus of limited specificity. Here, we discuss the generation of metabolic 'precision tools' to study particular subtypes of the glycome. A chemical biology tactic termed bump-and-hole engineering generates mutant glycosyltransferases that specifically accommodate bioorthogonal monosaccharides as an enabling technique of glycobiology. We review the groundbreaking discoveries that have led to applying the tactic in the living cell and the implications in the context of current developments in mass spectrometry glycoproteomics.
The use of mass spectrometry has been increasing in biological sciences over the last few decades as the technology has been getting more sensitive and specialised. From intact analysis of proteins to quantification of peptides or small molecules, the use of mass spectrometry continues to increase as it spreads through more disciplines in biological sciences. Here, four different questions were attempted to be answered starting with the less complex of identifying hydroxysterols in plasma as a method of identifying changes in the blood stream. Within the plasma ratios were created of 24S- and 27-hydroxycholesterol with cholesterol acids to create a relative quantification technique for analysis. The complexity increases with the identification of proteins involved in the synthesis of cholesterol and its metabolites in rat brain followed by identifying potential proteins involved in the synthesis of a UV absorbing molecule shinorine in the cyanobacteria Anabaena variabilis. Both proteomics analyses were able to identify some of the proteins involved in their respective pathways, while the cyanobacteria also quantified the levels of shinorine that were present by HPLC. While a few proteins were identified, it becomes obvious that more separation and sensitivity is needed to identify more of the pathways as they are not in high abundance in their respective proteomes. The final analysis performed was to understand the interaction of the protein STAT3β with a known and published inhibitor, STATTIC. Using in-gel digest, STATTIC was found to bind to a cysteine residue away from the SH2 domain of STAT3β. Within each question, mass spectrometry has been shown to be a valuable tool, however it is also shown to have drawbacks. It is shown most valuable when used to complement other techniques such as NMR, SDS-PAGE and Western blots, fluorescence assays and HPLC.
Changes in glycoprotein content, altered glycosylations and aberrant glycan structures are increasingly recognized as cancer hallmarks. Since breast cancer is one of the most common causes of cancer deaths in the world, it is highly urgent to find next/other reliable biomarkers for its initial diagnosis and to learn as much as possible about this disease. In this minireview, the applications of lectins for a screening of potential breast cancer biomarkers published during recent years are overviewed. The data provide a deeper insight into the use of modern strategies, technologies and scientific knowledge in glycoproteomic breast cancer research. Particular attention is concentrated on the using of lectin-based affinity techniques, applied independently or in combination most frequently with mass spectrometry, as an effective tool for targeting, separation and reliable identification of glycoprotein molecules. Individual procedures and lectins used in published glycoproteomic studies of breast cancer-related glycoproteins are discussed. Summarized approaches have the potential for use in diagnostic and predictive applications. Finally, the use of lectins is briefly discussed from a view of their future application in the analysis of glycoproteins in cancer.
Colorectal cancer associates with changed IgG glycosylation, but the alteration in specific subclass of IgG is unknown. Initially, we optimized five common IgG glycopeptides enrichment methods to acquire comprehensive profile of IgG glycopeptides. However, incomplete tryptic digestion of IgG occurred when using ordinary protease to protein ratio, which significantly impacted the final statistical analysis. Herein, we introduced a two-step enzymatic digestion, enabling the complete digestion of IgG glycopeptides and further improving the detection intensity of the target glycopeptides. In order to rapidly process and automatically integrate the MS data, we developed a simple and effective code by MATLAB. Following statistical analysis, we observed that IgG1_H3N4F1 and IgG1_H3N4 were substantially increased in CRC, while IgG1_H5N5F1, IgG1_H5N4F1S1 and IgG2_H5N4F1 were markedly decreased. Further evaluation of the diagnostic performance showed they all obtained a fair performance in discriminating the patients from the normal. In terms of the glycan features, it demonstrated that CRC progression associates with the increased agalactosylation, and the decreased digalactosylation and galactosylation per antenna on diantennay glycans of IgG1 and IgG2. Concurrently, the decreased sialylation of IgG1 was strongly correlated with CRC. Moreover, analysis of tumor-specific glycosylation showed that the alterations of IgG glycosylation were more significant in colon cancer, and no obvious difference between colon and rectal cancer. This study comprehensively optimized the glycopeptide enrichment methods, evaluated the enzymatic digestion effect, and explored the association between CRC progression and subclass-specific glycosylation.
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.
Background:
N-linked glycoprotein is a highly interesting class of proteins for clinical and biological research. The large-scale characterization of N-linked glycoproteins accomplished by mass spectrometry-based glycoproteomics has provided valuable insights into the interdependence of glycoprotein structure and protein function. However, these studies focused mainly on the analysis of specific sample type, and lack the integration of glycoproteomic data from different tissues, body fluids or cell types.
Methods:
In this study, we collected the human glycosite-containing peptides identified through their de-glycosylated forms by mass spectrometry from over 100 publications and unpublished datasets generated from our laboratory. A database resource termed N-GlycositeAtlas was created and further used for the distribution analyses of glycoproteins among different human cells, tissues and body fluids. Finally, a web interface of N-GlycositeAtlas was created to maximize the utility and value of the database.
Results:
The N-GlycositeAtlas database contains more than 30,000 glycosite-containing peptides (representing > 14,000 N-glycosylation sites) from more than 7200 N-glycoproteins from different biological sources including human-derived tissues, body fluids and cell lines from over 100 studies.
Conclusions:
The entire human N-glycoproteome database as well as 22 sub-databases associated with individual tissues or body fluids can be downloaded from the N-GlycositeAtlas website at http://nglycositeatlas.biomarkercenter.org.
Most serum proteins are N-linked glycosylated and therefore the glycoproteomic profiling of serum is essential for characterization of serum proteins. In this study, we profiled serum N-glycoproteome by our recently developed N-glycoproteomic method using solid-phase extraction of N-linked Glycans And Glycosite-containing peptides (NGAG) coupled with LC-MS/MS and site-specific glycosylation analysis using GPQuest software. Our data indicated that half of identified N-glycosites were modified by at least two glycans, with a majority of them were sialylated. Specifically, 3/4 of glycosites were modified by biantennary N-glycans and 1/3 of glycosites were modified by triantennary sialylated N-glycans. In addition, two novel atypical glycosites (with N-X-V motif) were identified and validated from albumin (ALB) and Alpha-1B-glycoprotein (A1BG). The widespread of these two glycosites among individuals were further confirmed by individual serum analyses.
The system-wide site-specific analysis of intact glycopeptides is crucial for understanding the exact functional relevance of protein glycosylation. Dedicated workflow with the capability to simultaneously characterize and quantify intact glycopeptides...
Glycomic and glycoproteomic analyses involve the characterization of oligosaccharides (glycans) conjugated to proteins. Glycans are produced through a complicated nontemplate driven process involving the competition of enzymes that extend the nascent chain. The large diversity of structures, the variations in polarity of the individual saccharide residues, and the poor ionization efficiencies of glycans all conspire to make the analysis arguably much more difficult than any other biopolymer. Furthermore, the large number of glycoforms associated with a specific protein site makes it more difficult to characterize than any post-translational modification. Nonetheless, there have been significant progress, and advanced separation and mass spectrometry methods have been at its center and the main reason for the progress. While glycomic and glycoproteomic analyses are still typically available only through highly specialized laboratories, new software and workflow is making it more accessible. This review focuses on the role of mass spectrometry and separation methods in advancing glycomic and glycoproteomic analyses. It describes the current state of the field and progress toward making it more available to the larger scientific community.
Introduction: Protein glycosylation is recognized as an important post-translational modification, with specific substructures having significant effects on protein folding, conformation, distribution, stability and activity. However, due to the structural complexity of glycans, elucidating glycan structure-function relationships is demanding. The fine detail of glycan structures attached to proteins (including sequence, branching, linkage and anomericity) is still best analysed after the glycans are released from the purified or mixture of glycoproteins (glycomics). The technologies currently available for glycomics are becoming streamlined and standardized and many features of protein glycosylation can now be determined using instruments available in most protein analytical laboratories.
Areas covered: This review focuses on the current glycomics technologies being commonly used for the analysis of the microheterogeneity of monosaccharide composition, sequence, branching and linkage of released N- and O-linked glycans that enable the determination of precise glycan structural determinants presented on secreted proteins and on the surface of all cells.
Expert commentary: Several emerging advances in these technologies enabling glycomics analysis are discussed. The technological and bioinformatics requirements to be able to accurately assign these precise glycan features at biological levels in a disease context are assessed.
Acetone precipitation was evaluated as a rapid, simple, low-cost and efficient method for the selective purification of O-glycopeptides from enzymatic digests of glycoproteins. Ovalbumin (OVA), human and bovin alpha-1-acid glycoprotein (hAGP and bAGP), human apolipoprotein C-III (APO-C3) and recombinant human erythropoietin (rhEPO) were used to obtain enzymatic digests with a broad and varied set of peptides, N-glycopeptides and O-glycopeptides. After digestion and before capillary electrophoresis mass spectrometry (CE-MS) analysis, the amount of ice-cold acetone added to the digests was optimized to maximize recoveries of O-glycopeptides. Furthermore, the different behaviour of peptides, N- and O-glycopeptides was explained by studying with multivariate data analysis methods the influence of several physicochemical parameters and properties related to their composition and structure. Principal component analysis (PCA) and, afterwards, partial least squares discriminant analysis (PLS-DA) were used to identify the most significant variables and their importance to differentiate between peptides, N-glycopeptides and O-glycopeptides, or within these classes. This information was useful to understand precipitation of these compounds after addition of acetone, and for the selection of the optimal conditions for purification of specific O-glycopeptide biomarkers. Special attention was paid to O126-glycopeptide glycoforms of rhEPO, because of their applicability in biopharmaceutical quality control and doping analysis.
This chapter focuses on the common molecular variants and degradation pathways of protein therapeutics generated under normal manufacturing and storage conditions. The analytical characterization of biologics is intended to provide an understanding of the structures and properties of molecular variants, which can be generated during cell culture, protein purification, storage, and in vivo after administration. Mass spectrometry (MS) has long been a major tool supporting development of each of these process areas with comprehensive information that few other techniques can match. Disulfide bond formation is an important posttranslational modification that helps to stabilize protein conformation. Within eukaryotes, glycosylation represents one of the most complex posttranslational modifications observed. One of the most common chemical modifications observed on protein therapeutics is oxidation. Oxidation can occur at any point during manufacture and storage, and stressors that may induce oxidation in protein therapeutics are numerous.
Protein N-glycosylation is essential for mammalian cell survival and is well-known to be involved in many biological processes. Aberrant glycosylation is directly related to human disease including cancer and infectious diseases. Global analysis of protein N-glycosylation will allow a better understanding of protein functions and cellular activities. Mass spectrometry (MS)-based proteomics provides a unique opportunity to site-specifically characterize protein glycosylation on a large scale. Due to the complexity of biological samples, effective enrichment methods are critical prior to MS analysis. Here, we compared two lectin-independent methods to enrich glycopeptides for the global analysis of protein N-glycosylation by MS. The first boronic acid-based enrichment (BA) method benefits from the universal and reversible interactions between boronic acid and sugars; the other method utilizes metabolic labeling and click chemistry (MC) to incorporate a chemical handle into glycoproteins for future affinity enrichment. We comprehensively compared the performance of the two methods in the identification and quantification of glycoproteins in statin-treated liver cells. Based on the current results, the BA method is more universal in enriching glycopeptides, while with the MC method, cell surface glycoproteins were highly enriched, and the quantification results appear to be more dynamic because only the newly-synthesized glycoproteins were analyzed. In addition, we normalized the glycosylation site ratios by the corresponding parent protein ratios to reflect the real modification changes. In combination with MS-based proteomics, effective enrichment methods will vertically advance protein glycosylation research.
Protein glycosylation is of increasing interest due to its important roles in protein function and aberrant expression with disease. Characterizing protein glycosylation remains analytically challenging due to its low abundance, ion suppression issues, and microheterogeneity at glycosylation sites, especially in complex samples such as human plasma. In this study, the utility of three common N-linked glycopeptide enrichment techniques is compared using human plasma. By analysis on an LTQ-Orbitrap Elite mass spectrometer, electrostatic repulsion hydrophilic interaction liquid chromatography using strong anion exchange solid-phase extraction (SAX-ERLIC) provided the most extensive N-linked glycopeptide enrichment when compared with multilectin affinity chromatography (M-LAC) and Sepharose-HILIC enrichments. SAX-ERLIC enrichment yielded 191 unique glycoforms across 72 glycosylation sites from 48 glycoproteins, which is more than double that detected using other enrichment techniques. The greatest glycoform diversity was observed in SAX-ERLIC enrichment, with no apparent bias toward specific glycan types. SAX-ERLIC enrichments were additionally analyzed by an Orbitrap Fusion Lumos mass spectrometer to maximize glycopeptide identifications for a more comprehensive assessment of protein glycosylation. In these experiments, 829 unique glycoforms were identified across 208 glycosylation sites from 95 plasma glycoproteins, a significant improvement from the initial method comparison and one of the most extensive site-specific glycosylation analysis in immunodepleted human plasma to date. Data are available via ProteomeXchange with identifier PXD005655.
High performance liquid chromatography (HPLC) is currently one of the most powerful analytical tools that has revolutionized the field of proteomics. Formerly known as high pressure liquid chromatography, this technique was introduced in the early 1960s to improve the efficiency of liquid chromatography separations using small stationary phase particles packed in columns. Since its introduction, continued advancements in column technology, development of different stationary phase materials and improved instrumentation has allowed the full potential of this technique to be realized. The various modes of HPLC in combination with mass spectrometry has evolved into the principal analytical technique in proteomics. It is now common practice to combine different types of HPLC in a multidimensional workflow to identify and quantify peptides and proteins with high sensitivity and resolution from limited amounts of samples. More recently, the introduction of Ultra High Performance Liquid Chromatography (UHPLC) has further raised the level of performance of this technique with significant increases in resolution, speed and sensitivity. The number of applications of HPLC and UHPLC in proteomics has been rapidly expanding and will continue to be a pivotal analytical technique. The aim of the following sections is to familiarize the beginner with the various HPLC methods routinely used in proteomics and provide sufficient practical knowledge regarding each of them to develop a separation and analytical protocol.
This review covers the basics and some applications of methodologies for the analysis of glycoprotein glycans. Analytical techniques used for glycoprotein glycans, including liquid chromatography (LC), capillary electrophoresis (CE), mass spectrometry (MS), and high-throughput analytical methods based on microfluidics, were described to supply the essentials about biopharmaceutical and biomarker glycoproteins. We will also describe the MS analysis of glycoproteins and glycopeptides as well as the chemical and enzymatic releasing methods of glycans from glycoproteins and the chemical reactions used for the derivatization of glycans. We hope the techniques have accommodated most of the requests from glycoproteomics researchers.
Lectin affinity chromatography (LAC) is an important enrichment technique in glycoproteomics analysis. In order to improve the effectiveness of enrichment, it is necessary to develop LAC materials with high specificity and efficiency. Herein, using oxidized dextran as the spacer, a silica-based concanavalin A material (SiO2-ODex Con A) was synthesized to enrich glycoproteins/glycopeptides. For comparison, the SiO2-Ald Con A material was synthesized using conventional (3-glycidoxypropyl) triethoxysilane (GPMS) as the initial spacer arm. The analytical merits of both Con A materials, such as non-specific adsorption, binding capacity and trapping efficiency, have been evaluated using ovalbumin. Under high performance liquid affinity chromatography (HPLAC) mode, the SiO2-ODex Con A material was highly effective in the enrichment of glycoproteins/glycopeptides attached to high-mannose-type and bi-antennary complex-type glycans. The promising potential of the SiO2-ODex Con A material was demonstrated by selective fractionation of glycoproteins from complex biological samples for glycosylation analysis.
Here we describe novel application areas in which hydrophilic interaction liquid chromatography (HILIC) has vast importance and utility, including peptides, glycopeptides, two-dimensional liquid chromatography in proteomics, oligonucleotides, lipids and lipidomics, glycan analysis, proteins, glycoproteins, phosphoproteins, and related biopolymers. We also describe some specific HILIC conditions that have proven useful for these classes of biologically active analytes and provide typical chromatograms from the recent literature, both scientific and commercial.
Protein glycosylation is a vital bio-process in organisms. Selective enrichment of glycopeptides before mass spectrometry (MS) plays an important role in proteomics owing to the low glycopeptides proportion and the suppression effect of the non-glycosylated peptides. There are several methods used for the enrichment of glycopeptides with their own merits and drawbacks. Among them, hydrophilic interaction liquid chromatography (HILIC) is increasingly applied to glycopeptides enrichment with its promising prospects. In this paper, a new strategy for the enrichment of glycopeptides using a commercial weak cation exchange chromatography (WCX) material under HILIC mode was developed. The influence of concentration of acetonitrile (ACN) and pH value on the retention of tryptic fetuin digests on WCX were studied. Results indicated that non-glycosylated peptides on WCX micro-column were eluted prior to glycopeptides when the concentration of ACN gradually decreased. Furthermore, the hydropathy index of peptides contained in glycopeptides also affected the retention. The bigger the index was, the later the plycopeptides eluted. Meanwhile, the glycopeptides were retained more strongly at lower pH than higher pH. When using WCX to enrich glycopeptides, it was found that not only sialic acid containing glycopeptides but also neutral ones could be selectively enriched. In the enrichment of tryptic IgG digests, 25 glycopeptides were found by WCX while only 1 glycopeptides were found before enrichment. And when the tryptic fetuin digests were enriched by WCX, the glycopeptides numbers detected by MS increased to 39, in contrast to only 2 before enrichment. What's more, WCX can enrich glycopeptides even under relatively complex surrounding. 23 IgG glycopeptides and 26 fetuin glycopeptides could be enriched by WCX when they were respectively mixed with bovine serum albumin (BSA) at a molar ratio of 1:5. The results indicated that glycopeptides could be selectively enriched by WCX under HILIC mode. This is a new glycopeptides enrichment method, which expands the application of WCX and enriches the variety of HILIC material. © 2015 Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences.
Kay-Hooi Khoo graduated from the Department of Biochemistry, Imperial College, London, in 1989 and went on to do his Ph.D. studies with Professor Anne Dell at the same department. Specializing in mass spectrometry applications to solving glycan structures, he obtained his doctorate degree in 1993 and stayed for another year and a half at Imperial College as a Wellcome Prize Fellow, before moving to Colorado State Univeristy in 1994 for another postdoctoral fellowship. At Colorado State University, he focused on analyzing the mycobacterial cell wall-associated glycoconjugates, a work that was initiated during his Ph.D. studies in collaboration with Dr. Delphi Chatterjee and Professor Patrick Brennan of the Mycobacterial Research Laboratories at Colorado State University. In 1996, he was recruited as an associate research fellow to the Institute of Biological Chemistry, Academia Sinica, Taiwan, where he is now a full research fellow. Dr. Khoo’s research interests remain focused on applications of mass spectrometry to glycan analysis, but have expanded to include protein glycosylation, and more recently to glycomics, proteomics, and glycoproteomics. He is the director of the Core Facilities for Proteomics and Glycomics under the Taiwan National Research Program in Genomic Medicine and is engaged as participating investigator in the NIH Consortium for Functional Glycomics, and the HGPI of human proteome organisation (HUPO).
Glycoproteomic researches have been rapidly progressing in the last few years. Although intact glycopeptide analysis which obtains glycosite and corresponding glycan information simultaneously is still challenging, various studies, combining glycosylation enrichment methods and mass spectrometry techniques, have accumulated a considerable amount of protein glycosite data for either N- or O-glycosylation or O-GlcNAcylation. This review will majorly focus on recent research outputs in the field of glycosite exploration.
Hydrophilic interaction chromatography (HILIC) adsorbents have drawn increasing attention in recent years due to their high efficiency in N-glycopeptides enrichment. The hydrophilicity and binding capacity of HILIC adsorbents are crucial to the enrichment efficiency and mass spectrometry (MS) detection sensitivity of N-glycopeptides. Herein, magnetic nanoparticles coated with maltose-functionalized polyethyleneimine (Fe3O4-PEI-Maltose MNPs) were prepared by one-pot solvothermal reaction coupled with "click chemistry" and utilized for N-glycopeptides enrichment. Owing to the presence of hydrophilic and branched polyethyleneimine, the amount of immobilized disaccharide units was improved about four times. The N-glycopeptides capturing capacity was about 150mg/g (IgG/MNPs) and the MS detection limitation as low as 0.5fmol for IgG and 85% average enrichment recovery were feasibly achieved by using this hybrid magnetic adsorbent. Finally, 1237 unique N-glycosylation sites and 1567 unique N-glycopeptides from 684 N-glycoproteins were reliably characterized from 60μg protein sample extracted from mouse liver. Therefore, this maltose-functionalized polyethyleneimine coated adsorbent can play a promising role in highly efficient N-glycopeptides enrichment for glycoproteomic analyses of complex protein samples.
This chapter discusses the use of hydrophilic interaction chromatography (HILIC) for analysis of biochemical compounds. Some compounds such as amino acids, carbohydrates, nucleobases, nucleosides, oligonucleotides, peptides, phospholipids, and proteins are of interest as components in pharmaceutical or food products. The chapter focuses on application of HILIC to separations of these types of compounds in problems of biochemical and related interests. Many recent glycan analysis applications have employed zwitterionic- HILIC (ZIC-HILIC) or neutral amide stationary phases. Other types of HILIC columns have been used for certain applications, but it seems that the field is moving toward the former two column types as first-line choices. Fluorescence detection of derivatized glycans or mass spectrometric detection is clearly the mode of choice, with mass spectrometry (MS) providing superior data for structural characterization. Amide or zwitterionic phases have also become popular for other biomolecules such as nucleobases, nucleosides, nucleotides, oligonucleotides, amino acids, and peptides.
Plasma glycoproteins and extracellular vesicles represent excellent sources of disease biomarkers, but laboratory detection of these circulating structures are limited by their relatively low abundance in complex biological fluids. While intensive research has led to the development of effective methods for the enrichment and isolation of either plasma glycoproteins or extracellular vesicles from clinical materials, at present it is not possible to enrich both structures simultaneously from individual patient sample, a method that affords the identification of biomarker combinations from both entities for the prediction of clinical outcomes will be clinical useful. We have therefore developed an enrichment method for use in mass spectrometry-based proteomic profiling that couples prolonged ultracentrifugation (PUC) with electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) [PUC-ERLIC], to facilitate the recovery of both glycoproteins and extracellular vesicles from non-depleted human plasma. Following PUC, plasma glycoproteins and extracellular vesicles were concentrated as a yellow suspension, and simultaneous analyses of low abundant secretory and vesicular glycoproteins was achieved in a single LC-MS/MS run. Using this systematic PUC-ERLIC approach, we identified a total of 127 plasma glycoproteins at a high level of confidence (FDR ≤ 1%), including 48 glycoproteins with concentrations ranging from pg to ng/mL. The novel enrichment method we report should facilitate future human plasma-based proteome and glycoproteome that will identify novel biomarkers, or combinations of secreted and vesicle-derived biomarkers, that can be used to predict clinical outcomes in human patients.
Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
Pancreatic cancer is the fourth leading cause of cancer-related death in the United States, with a 5-year survival rate of less than 4%. Effective early detection and screening are currently not available, and tumors are typically diagnosed at a late stage, frequently after metastasis. Existing clinical markers of pancreatic cancer lack specificity, as they are also found in inflammatory diseases of the pancreas and biliary tract. In the work described here, naturally occurring glycoproteins were enriched by using lectin affinity chromatography and then further resolved by nonporous reversed-phase chromatography. Glycoprotein microarrays were then printed and probed with a variety of lectins to screen glycosylation patterns in sera from normal, chronic pancreatitis, and pancreatic cancer patients. Ten normal, 8 chronic pancreatitis, and 6 pancreatic cancer sera were investigated. Data from the glycoprotein microarrays were analyzed using bioinformatics approaches including principal component analysis (PCA) and hierarchical clustering (HC). Both normal and chronic pancreatitis sera were found to cluster close together, although in two distinct groups, whereas pancreatic cancer sera were significantly different from the other two groups. Both sialylation and fucosylation increased as a function of cancer on several proteins including Hemopexin, Kininogen-1, Antithrombin-III, and Haptoglobin-related protein, whereas decreased sialylation was detected on plasma protease C1 inhibitor. Target alterations on glycosylations were verified by lectin blotting experiments and peptide mapping experiments using microLC-ESI-TOF. These altered glycan structures may have utility for the differential diagnosis of pancreatic cancer and chronic pancreatitis and identify critical differences between biological samples from patients with different clinical conditions.
The three-dimensional structure of the lectin concanavalin A (Con A) has been determined at 2.0-A resolution by x-ray diffraction analysis. The protomers are ellipsoidal domes of dimensions 42 times 40 times 39 A. Folding of the polypeptide backbone is dominated by the presence of two antiparallel pleated sheets, a twisted sheet of seven strands passing through the center of the molecule and a bowed sheet of six strands which forms the back surface of the monomer. Manganese and calcium ions bind to the protein at adjoining sites to form a binuclear complex of two octahedra sharing a common edge. The ligands for each metal ion are four groups from the NH2-terminal region of the protein and 2 water molecules. The binding site for the inhibitor beta-(o-iodophenyl)-D-glucopyranoside is in a deep cavity which contains distinct hydrophobic and hydrophilic binding subsites. Studies of the binding of beta-(o-iodophenyl)-D-glucopyranoside to Con A in the crystalline state and in solution have indicated that the binding behavior of the protein is somewhat different in the two states.
Evidence is presented for the existence of specific receptors for alpha-fetoprotein on the surface of MCF-7 human breast cancer cells. At 4 degrees C, the binding of alpha-fetoprotein to these cells displayed a biphasic saturation curve. Scatchard analysis revealed the presence of at least two binding sites with dissociation constants of 4.5 X 10(-9) M (2,000 sites/cell) and 1.3 X 10(-8) M (135,000 sites/cell), respectively. Binding was inhibited by 85% in the presence of a 5,000-fold excess of unlabeled alpha-fetoprotein and by 50% with the same excess of serum albumin. Competition by other serum proteins was not significant. At 37 degrees C, alpha-fetoprotein was endocytosed and the uptake curve reached a plateau after 3-4 hours of incubation.
Complement factor H (FH) and factor-H-like protein 1 (FHL-1) are human plasma proteins with regulatory functions in the alternative pathway of complement activation. FH and FHL-1 are organized in repetitive elements termed short consensus repeats (SCRs) and the seven SCRs of FHL-1 are identical with the N-terminal domain of the 20 SCRs of FH. The fourth SCR of both proteins (SCR 4) includes the sequence Arg-Gly-Asp (RGD), a motif that is responsible for the major adhesive activity of matrix proteins like fibronectin. A synthetic hexapeptide with the sequence ERGDAV derived from the RGD domain of FH/FHL-1 interferes with cell attachment to a fibronectin matrix. Although the identical motif is present in both FH and FHL-1, only FHL-1 acts as a matrix for cell spreading and attachment, thus the two proteins differ in function. The adhesive activity of FHL-1 is localized to the RGD-containing SCR 4 by the use of recombinant fragments. All three analysed anchorage-dependent cell lines (CCl64, C32 and MRC-5) adhere to an FHL-1 matrix. The use of synthetic peptides in competition assays, on either FHL-1-derived or fibronectin matrices, shows that the cellular receptors binding to the FH/FHL-1-derived RGD motif are related to or identical with integrin receptors which interact with fibronectin. The identification of a functional adhesive domain in the FH/FHL-1 sequence demonstrates, at least for FHL-1, a role in cell attachment and adhesion.
In serum, human paraoxonase/arylesterase (PON1) is found exclusively associated with high density lipoprotein (HDL) and contributes to its antiatherogenic properties by inhibiting low density lipoprotein (LDL) oxidation. Difficulties in purifying PON1 from apolipoprotein A-I (apoA-I) suggested that PON1's association with HDL may occur through a direct binding between these 2 proteins. An unusual property of PON1 is that the mature protein retains its hydrophobic N-terminal signal sequence. By expressing in vitro a mutant PON1 with a cleavable N-terminus, we demonstrate that PON1 associates with lipoproteins through its N-terminus by binding phospholipids directly rather than binding apoA-I. Nonetheless, apoA-I stabilized arylesterase activity more than did phospholipid alone, apoA-II, or apoE. Consequently, we studied the role of apoA-I in PON1 expression and HDL association in mice genetically deficient in apoA-I. Though present in HDL fractions at decreased levels, PON1 arylesterase activity was less stable than in control mice. Furthermore, PON1 could be competitively removed from HDL by phospholipids, suggesting that PON1's retained N-terminal peptide allows transfer of the enzyme between phospholipid surfaces. Thus, our data suggest that PON1 is stabilized by apoA-I, and its binding to HDL and physiological distribution are dependent on the direct binding of the retained hydrophobic N-terminus to phospholipids optimally presented in association with apoA-I.
Of over 20 nucleated cell lines we have examined to date, human H2 glioblastoma cells have turned out to be the most resistant to complement-mediated cytolysis in vitro. H2 cells expressed strongly the membrane attack complex inhibitor protectin (CD59), moderately CD46 (membrane cofactor protein) and CD55 (decay-accelerating factor), but no CD35 (complement receptor 1). When treated with a polyclonal anti-H2 Ab, anti-CD59 mAb, and normal human serum, only 5% of H2 cells became killed. Under the same conditions, 70% of endothelial-like EA.hy 926 cells and 40% of U251 control glioma cells were killed. A combined neutralization of CD46, CD55, and CD59 increased H2 lysis only minimally, demonstrating that these complement regulators are not enough to account for the resistance of H2 cells. After treatment with Abs and serum, less C5b-9 was deposited on H2 than on U251 and EA.hy 926 cell lines. A reason for the exceptional resistance of H2 cells was revealed when RT-PCR and protein biochemical methods showed that the H2 cells, unlike the other cell lines tested, actively produced the soluble complement inhibitors factor H and factor H-like protein 1. H2 cells were also capable of binding human factor H from the fluid phase to their cell surface and promoted the cleavage of C3b to its inactive form iC3b more efficiently than U251 and EA.hy 926 cells. In accordance, anti-factor H mAbs enhanced killing of H2 glioblastoma cells. Taken together, our results show that production and binding of factor H and factor H-like protein 1 is a novel mechanism that these malignant cells utilize to escape complement-mediated killing.
N- and O-oligosaccharide variants on glycoproteins (glycoforms) can lead to alterations in protein activity or function that may manifest themselves as overt disease.
This review summarizes those diseases that are known to be the result of an inherited or acquired glycoprotein oligosaccharide structural alteration and that are diagnosed in blood or urine by chemical characterization of that oligosaccharide alteration.
The biochemical synthesis steps and catabolic pathways important in determining glycoprotein function are outlined with emphasis on alterations that lead to modified function. Clinical and biochemical aspects of the diagnosis are described for inherited diseases such as I-cell disease, congenital disorders of glycosylation, leukocyte adhesion deficiency type II, hereditary erythroblastic multinuclearity with a positive acidified serum test, and Wiskott-Aldrich syndrome. We also review the laboratory use of measurements of glycoforms related to acquired diseases such as alcoholism and cancer.
Identification of glycoprotein glycoforms is becoming an increasingly important laboratory contribution to the diagnosis and management of human diseases as more diseases are found to result from glycan structural alterations.
Chemical tools have proven indispensable for studies in glycobiology. Synthetic oligosaccharides and glycoconjugates provide
materials for correlating structure with function. Synthetic mimics of the complex assemblies found on cell surfaces can modulate
cellular interactions and are under development as therapeutic agents. Small molecule inhibitors of carbohydrate biosynthetic
and processing enzymes can block the assembly of specific oligosaccharide structures. Inhibitors of carbohydrate recognition
and biosynthesis can reveal the biological functions of the carbohydrate epitope and its cognate receptors. Carbohydrate biosynthetic
pathways are often amenable to interception with synthetic unnatural substrates. Such metabolic interference can block the
expression of oligosaccharides or alter the structures of the sugars presented on cells. Collectively, these chemical approaches
are contributing great insight into the myriad biological functions of oligosaccharides.
The ability of physicians to effectively treat and cure cancer is directly dependent on their ability to detect cancers at their earliest stages. Proteomic analyses of early-stage cancers have provided new insights into the changes that occur in the early phases of tumorigenesis and represent a new resource of candidate biomarkers for early-stage disease. Studies that profile proteomic patterns in body fluids also present new opportunities for the development of novel, highly sensitive diagnostic tools for the early detection of cancer.
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.
Serum specimens from 35 patients with laryngeal cancer (10 at stage II; 12 at stage III; 1.3 at stage IV) were obtained before therapy was initiated. Concentrations of bound sialic acid (BSA), free sialic acid (FSA), and alpha-1-acid glycoprotein (AAG) were compared to those in serum specimens from 34 healthy controls. The mean levels of serum BSA and AAG were significantly increased in the laryngeal cancer patients versus the controls; there was no significant difference in serum FSA levels between the patients and controls. Mean serum BSA and AAG levels were lowest in the control group and highest in the stage IV patients. As the stage of the cancer advanced, progressively higher levels of serum BSA and AAG were observed. The results indicate that serum BSA and AAG, but not FSA, show correlation with the stage of laryngeal carcinoma.
In this study, we show that recombinant human histidine-rich glycoprotein (HRGP) has potent antiangiogenic properties as judged from effects on a syngeneic tumor model in C57/bl6 mice. Growth of fibrosarcoma, a very aggressive tumor, was reduced by >60% by HRGP treatment, and tumor angiogenesis was dramatically decreased. Treatment with HRGP led to increased apoptosis and reduced proliferation in the tumors. In contrast, HRGP did not affect apoptosis or DNA synthesis in endothelial cells or tumor cells in vitro. The mechanism of action of HRGP involves rearrangement of focal adhesions and decreased attachment of endothelial cells to vitronectin and, as a consequence, reduced endothelial cell migration. By using truncated versions of HRGP, we demonstrate that the isolated 150 amino acid-residue His/Pro-rich domain, which is also released by spontaneous proteolysis from purified HRGP, mediates the inhibitory effect on chemotaxis. Moreover, the His/Pro-rich domain must be released from HRGP to exert its effect. This study shows for the first time inhibitory effects of HRGP on tumor vascularization in vivo, thus providing proof of concept that HRGP is an angiogenesis inhibitor.
The Golgi enzyme β1,6 N-acetylglucosaminyltransferase V (Mgat5) is up-regulated in carcinomas and promotes the substitution
of N-glycan with poly N-acetyllactosamine, the preferred ligand for galectin-3 (Gal-3). Here, we report that expression of
Mgat5 sensitized mouse cells to multiple cytokines. Gal-3 cross-linked Mgat5-modified N-glycans on epidermal growth factor
and transforming growth factor–β receptors at the cell surface and delayed their removal by constitutive endocytosis. Mgat5
expression in mammary carcinoma was rate limiting for cytokine signaling and consequently for epithelial-mesenchymal transition,
cell motility, and tumor metastasis. Mgat5 also promoted cytokine-mediated leukocyte signaling, phagocytosis, and extravasation
in vivo. Thus, conditional regulation of N-glycan processing drives synchronous modification of cytokine receptors, which
balances their surface retention against loss via endocytosis.
Both aging and diabetes are characterized by the formation of advanced glycation end products (AGEs). Both exhibit other similarities including deficits in wound healing that are associated with higher rates of fibroblast apoptosis. In order to investigate a potential mechanism for enhanced fibroblast apoptosis in diabetes and aged individuals, experiments were carried out to determine whether the predominant advanced glycation end product in skin, N-epsilon-(carboxymethyl) lysine (CML)-collagen, could induce fibroblast apoptosis. In vivo experiments established that CML-collagen but not unmodified collagen induced fibroblast apoptosis and that apoptosis was dependent upon caspase-3, -8, and -9 activity. In vitro experiments demonstrated that CML-collagen but not control collagen induced a time- and dose-dependent increase in fibroblast apoptosis. By use of blocking antibodies, apoptosis was shown to be mediated through receptor for AGE signaling. AGE-induced apoptosis was largely dependent on the effector caspase, caspase-3, which was activated through both cytoplasmic (caspase-8-dependent) and mitochondrial (caspase-9) pathways. CML-collagen had a global effect of enhancing mRNA levels of pro-apoptotic genes that included several classes of molecules including ligands, receptors, adaptor molecules, mitochondrial proteins, and others. However, the pattern of expression was not identical to the pattern of apoptotic genes induced by tumor necrosis factor alpha.
Although mass spectrometry has become a powerful tool for the functional analysis of biological systems, complete proteome characterization cannot yet be achieved. Instead, the sheer complexity of living organisms demands fractionation of cellular extracts to enable more targeted analyses. Here, we introduce the concept of "fluorous proteomics," whereby specific peptide subsets from samples of biological origin are tagged with perfluorinated moieties and subsequently enriched by solid-phase extraction over a fluorous-functionalized stationary phase. This approach is extremely selective, yet can readily be tailored to enrich different subsets of peptides. Additionally, this methodology overcomes many of the limitations of traditional bioaffinity-based enrichment strategies, while enabling new affinity enrichment schemes impossible to implement with bioaffinity reagents. The potential of this methodology is demonstrated by the facile enrichment of peptides bearing particular side-chain functionalities or post-translational modifications from tryptic digests of individual proteins as well as whole cell lysates.
The use of mass spectrometry as a proteomics tool is poised to revolutionize early disease diagnosis and biomarker identification. Unfortunately, before standard supervised classification algorithms can be employed, the "curse of dimensionality" needs to be solved. Due to the sheer amount of information contained within the mass spectra, most standard machine learning techniques cannot be directly applied. Instead, feature selection techniques are used to first reduce the dimensionality of the input space and thus enable the subsequent use of classification algorithms. This paper examines feature selection techniques for proteomic mass spectrometry.
This study examines the performance of the nearest centroid classifier coupled with the following feature selection algorithms. Student-t test, Kolmogorov-Smirnov test, and the P-test are univariate statistics used for filter-based feature ranking. From the wrapper approaches we tested sequential forward selection and a modified version of sequential backward selection. Embedded approaches included shrunken nearest centroid and a novel version of boosting based feature selection we developed. In addition, we tested several dimensionality reduction approaches, namely principal component analysis and principal component analysis coupled with linear discriminant analysis. To fairly assess each algorithm, evaluation was done using stratified cross validation with an internal leave-one-out cross-validation loop for automated feature selection. Comprehensive experiments, conducted on five popular cancer data sets, revealed that the less advocated sequential forward selection and boosted feature selection algorithms produce the most consistent results across all data sets. In contrast, the state-of-the-art performance reported on isolated data sets for several of the studied algorithms, does not hold across all data sets.
This study tested a number of popular feature selection methods using the nearest centroid classifier and found that several reportedly state-of-the-art algorithms in fact perform rather poorly when tested via stratified cross-validation. The revealed inconsistencies provide clear evidence that algorithm evaluation should be performed on several data sets using a consistent (i.e., non-randomized, stratified) cross-validation procedure in order for the conclusions to be statistically sound.
Despite recent advances in our understanding of the significance of the protein glycosylation, the throughput of protein glycosylation analysis is still too low to be applied to the exhaustive glycoproteomic analysis. Aiming to elucidate the N-glycosylation of murine epidermis and dermis glycoproteins, here we used a novel approach for focused proteomics. A gross N-glycan profiling (glycomics) of epidermis and dermis was first elucidated both qualitatively and quantitatively upon N-glycan derivatization with novel, stable isotope-coded derivatization reagents followed by MALDI-TOF(/TOF) analysis. This analysis revealed distinct features of the N-glycosylation profile of epidermis and dermis for the first time. A high abundance of high mannose type oligosaccharides was found to be characteristic of murine epidermis glycoproteins. Based on this observation, we performed high mannose type glycoform-focused proteomics by direct tryptic digestion of protein mixtures and affinity enrichment. We identified 15 glycoproteins with 19 N-glycosylation sites that carry high mannose type glycans by off-line LC-MALDI-TOF/TOF mass spectrometry. Moreover the relative quantity of microheterogeneity of different glycoforms present at each N-glycan binding site was determined. Glycoproteins identified were often contained in lysosomes (e.g. cathepsin L and gamma-glutamyl hydrolase), lamellar granules (e.g. glucosylceramidase and cathepsin D), and desmosomes (e.g. desmocollin 1, desmocollin 3, and desmoglein). Lamellar granules are organelles found in the terminally differentiating cells of keratinizing epithelia, and desmosomes are intercellular junctions in vertebrate epithelial cells, thus indicating that N-glycosylation of tissue-specific glycoproteins may contribute to increase the relative proportion of high mannose glycans. The striking roles of lysosomal enzymes in epidermis during lipid remodeling and desquamation may also reflect the observed high abundance of high mannose glycans.
Both the urinary proteome and its glycoproteome can reflect human health status, and more directly, functions of kidney and urinary tracts. Because the high abundance protein albumin is not N-glycosylated, the urine N-glycoprotein enrichment procedure could deplete it, and urine proteome could thus provide a more detailed protein profile in addition to glycosylation information especially when albuminuria occurs in some kidney diseases. In terms of describing the details of urinary proteins, the urine glycoproteome is even a better choice than the proteome itself. Pooled urine samples from healthy volunteers were collected and acetone-precipitated for proteins. N-Linked glycoproteins enriched with concanavalin A affinity purification were separated and analyzed by SDS-PAGE-reverse phase LC/MS/MS or two-dimensional LC/MS/MS. A total of 225 urinary proteins were identified based on two-hit criteria with reliability over 97% for each peptide. Among these proteins, 94 were identified in previous urine proteome works, 150 were annotated as glycoproteins in Swiss-Prot, and 43 were predicted as glycoproteins by NetNGlyc 1.0. A number of known biomarkers and disease-related glycoproteins were identified. Because changes in protein quantity or the glycosylation status can lead to changes in the concanavalin A-captured glycoprotein profile, specific urine glycoproteome patterns might be observed for specific pathological conditions as multiplex urinary biomarkers. Knowledge of the urine glycoproteome is important in understanding kidney and body function.
The application of mass spectrometry to identify disease biomarkers in clinical fluids like serum using high throughput protein expression profiling continues to evolve as technology development, clinical study design, and bioinformatics improve. Previous protein expression profiling studies have offered needed insight into issues of technical reproducibility, instrument calibration, sample preparation, study design, and supervised bioinformatic data analysis. In this overview, new strategies to increase the utility of protein expression profiling for clinical biomarker assay development are discussed with an emphasis on utilizing differential lectin-based glycoprotein capture and targeted immunoassays. The carbohydrate binding specificities of different lectins offer a biological affinity approach that complements existing mass spectrometer capabilities and retains automated throughput options. Specific examples using serum samples from prostate cancer and hepatocellular carcinoma subjects are provided along with suggested experimental strategies for integration of lectin-based methods into clinical fluid expression profiling strategies. Our example workflow incorporates the necessity of early validation in biomarker discovery using an immunoaffinity-based targeted analytical approach that integrates well with upstream discovery technologies.
There are a limited number of noninvasive methods available for the monitoring of neoplastic disease in the central nervous system. The goal of our study was to find reliable markers that could be used for disease monitoring as well as to identify new targets for the therapeutic intervention for malignant astrocytoma (WHO grades 3 and 4).
We employed proteomic techniques to identify secreted proteins in the cerebrospinal fluid that were specific to patients with malignant astrocytoma.
Among 60 cerebrospinal fluid samples of patients with various central nervous system diseases, attractin was consistently found to be elevated in the samples of patients with malignant astrocytoma. To independently validate these results, we examined attractin expression in a new set of 108 normal and tumoral brain tissue specimens and found elevated expression in 97% of malignant astrocytomas, with the highest levels in grade 4 tumors. Using immunohistochemistry, we further showed that attractin is produced and secreted by the tumor cells. Finally, we showed that cerebrospinal fluid from brain tumor patients induces glioma cell migration and that attractin is largely responsible for this promigratory activity.
Our results find attractin to be a reliable secreted marker for high-grade gliomas. Additionally, our migration studies suggest that it may be an important mediator of tumor invasiveness, and thus, a potential target in future therapies.
The success attributed to identification and characterization of gel separated proteins by mass spectrometry (MS) is highly dependent on the percentage of an entire sequence covered by matching peptides derived from enzymatic digestion. Desalting and concentration of peptide mixtures on reversed‐phase (RP) microcolumns prior to mass spectrometric analysis have resulted in increased signal‐to‐noise ratio and sensitivity, and consequently higher sequence coverage. A large proportion of peptides, however, remains undetected by MS presumably because they are lost during sample preparation on microcolumns, or are suppressed in the ionization process. We report here the use of graphite powder packed in constricted GELoader tips as an alternative to RP microcolumns for desalting and concentration of peptide mixtures prior to MS. Such columns are able to retain small and/or hydrophilic peptides that can be lost when using RP microcolumns. In addition, we show that samples contaminated with small biological polymers can readily be analyzed using graphite powder rather than RP microcolumns, since the polymer molecules bind strongly to graphite and are not eluted with the peptides.
Assay of the serum α-1-acid glycoprotein (SAGP) produced by the liver in response to many disease states is shown to reflect prognosis and monitor progress in patients with early, recurrent and disseminated breast cancer. Eighteen of 26 patients with localized breast cancer and positive bone scans had elevated SAGP when first seen, but within 5 months of treatment 5 further patients with positive bone scans developed an elevated SAGP. Furthermore, an initally abnormal SAGP. evel became normal within 5 months in 5 of 6 patients with negative bone scans. While elevated SAGP in breast cancer correlates with positive bone scans, a series of normal values may indicate patients without early haematogenous dissemination of their disease.
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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.
Given the high specificity of plant lectins, it is not surprising that their immobilized forms are more and more frequently used for affinity-based analysis of glycoproteins. Common lectin-immobilization techniques range from reversible, non-covalent attachments to covalent immobilization onto a vast palette of substrates (e.g., agarose, silica, and polymeric materials). This article gives an overview of recently reported strategies in lectin immobilization to different stationary phases in chromatography and electrophoresis in order to support lectin-affinity-interaction-based bioseparation methodologies.
The majority of serum proteins are glycosylated. When disease is present, subtle changes occur in this glycosylation. These changes could provide the basis for more sensitive and more discriminative clinical tests. In order to address this possibility, a review is given of serum protein glycosylation in liver disease, inflammation and cancer. It is concluded that liver disease is accompanied by reduced sialylation and increased glycan branching; whereas cancer is accompanied by increased sialylation and increased fucosylation. In inflammation, the type of glycosylation change observed seems to depend upon the disease studied. Glycoprotein analysis can already be used for diagnosis in a few clinical situations; however, further studies are required in most diseases to provide a more detailed picture of the glycosylation changes that are occurring. This situation will change with the increasing availability of simpler techniques for glycoprotein analysis. One such group of techniques are lectin-based methods. The usefulness of these methods for glycoprotein analysis and the suitability for analysing clinical specimens are discussed in detail.
Lectins from Anguilla anguilla, Artocarpus integrifolia, Canavalia ensiformis, Datora stramonium, Glycine max, Limax flavus, Ricinus communis and Triticum vulgaris were tested for their abilities to antagonize the binding of botulinum neurotoxin and tetanus toxin to rat brain membranes and to antagonize the ability of these toxins to block neuromuscular transmission in mouse phrenic nerve-hemidiaphragm preparations. Lectins from Limax flavus and Triticum vulgaris, both of which have affinity for sialic acid, were antagonists of the various serotypes of botulinum neurotoxin and tetanus toxin. When tested against the high affinity binding site for botulinum neurotoxin type B, the lectin from Limax flavus had a Ki of 3.1 x 10(-7) M and the lectin from Triticum vulgaris had a Ki of 3.75 x 10(-7) M. When tested against the high affinity binding site for tetanus toxin, the lectins from Limax flavus and Triticum vulgaris had Ki values of 1.5 x 10(-7) and 1 x 10(-6) M, respectively. In all cases the lectins behaved as competitive antagonists. In reverse experiments, neither botulinum toxin nor tetanus toxin was a very effective antagonist of lectin binding to brain membranes. Studies on isolated neuromuscular preparations showed that the lectin from Triticum vulgaris did not affect transmission at concentrations of 10(-6) to 10(-3) M, but at a concentration of 3 x 10(-5) M the lectin produced highly statistically significant antagonism of the neuromuscular blocking properties of botulinum neurotoxin types A, B, C, D, E and F as well as tetanus toxin. The lectin did not antagonize beta-bungarotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)
The purification of membrane-bound cellular receptors has relied heavily on lectin chromatography and exemplifies some special problems. First, because a typical receptor constitutes a miniscule fraction of solubilized membrane protein, large quantities of cells or tissues are often required to obtain purified protein. Lectin binding can be detected using either iodinated lectin or peroxidase-linked avidin and biotinylated lectins. Typical values for the stoichiometry of binding of purified glycoproteins to immobilized lectins are less than 20% of theoretical, reflecting both inactivation of the lectin by derivitization to the support and steric factors. It is possible to take advantage of the different binding properties of glycoproteins in a complex mixture. Lectin affinity chromatography is rarely sufficient by itself for the purification of a glycoprotein. One should consider this approach as part of a general strategy for protein purification. It has been particularly useful in situations in which a limited amount of protein is available and requires a multistep purification procedure.
The relationship between cancer and coagulation disorders is widely accepted. Such disorders can contribute to the metastatic spreading of the primary tumor. Aim of our study was to evaluate the alterations of thrombin-antithrombin III complex (TAT) measured by an ELISA plasma assay in a population of 78 patients suffering from various gastrointestinal tumors. We found high levels of TAT in 68.6% of the patients. Our data show that assay of TAT plasma levels may be a useful test in detecting early coagulation disorders in cancer patients.
Direct evidence was obtained for the existence of a specific high affinity alpha-fetoprotein (AFP)-binding protein in the cytosol of both MCF-7 human breast cancer cultured cells and primary breast cancer tissue from postmenopausal women using a nitrocellulose blotting assay. Scatchard analysis of the binding data for MCF-7 cells at 37 degrees C revealed the presence of a single class of AFP binding sites with an apparent Kd of 4.5 x 10(-8) M, and 75,000 binding sites per cell. All 9 primary breast cancer cytosols obtained from postmenopausal women also contained measureable levels of this specific AFP-binding protein. The number of AFP molecules specifically bound varied considerably between patients and ranged from 29-250 fmol per mg cytosol protein. Levels of AFP-binding protein levels and estrogen receptor measured in these same breast cancer cytosols showed a positive statistical correlation (r = 0.85). Taken together, the present evidence for the existence of a specific cytoplasmic AFP-binding protein in MCF-7 cells and previously reported evidence for de novo synthesis of free immunoreactive and bound nonimmunoreactive forms of cytoplasmic AFP by MCF-7 cells is consistent with the conclusion that most of the endogenous AFP synthesized in breast cancer cells is rapidly bound to specific cytoplasmic AFP-receptors, and that binding of AFP to these receptors masks its immunoreactivity.(ABSTRACT TRUNCATED AT 250 WORDS)
Alpha 1 acid glycoprotein, alpha 1 antitrypsin, haptoglobin, alpha 2 HS glycoprotein and prealbumin were studied in 44 patients with cancer of the head and neck and in a control group. The three primary glycoproteins were considerably elevated in the patients, particularly in stage IV. In the group of patients who died before 30 months after treatment the haptoglobin was significantly elevated compared to those who survived beyond this point. Only values for the alpha 1 acid glycoprotein greater than 150 mg/dl were a prognostic factor of similar precision to that of stage IV. This correlation could not be shown for the other glycoproteins.
Electrospray ionization mass spectrometry utilizing a single quadrupole on line with reversed-phase HPLC (LC/MS) enables the characterization of glycoproteins in a relatively short period of time. In this approach the protein is digested with a suitable protease and the peptides are separated by reversed-phase HPLC and detected by electrospray ionization mass spectrometry. The glycopeptides are initially observed as a cluster of negatively sloping ions in a contour plot of data from the LC/MS run (m/z vs retention time) or as a characteristics series of masses at different elution times. The search for a particular glycopeptide is based on previously known carbohydrate structures and on consensus glycosylation sites. Further structural information is obtainable with glycosidase digestion and LC/MS analysis. The mass shifts following glycosidase digestion allow further confirmation of the structure. This approach identifies the site of attachment of two hybrid glycoforms to the T11 tryptic peptide in a reversed-phase tryptic map of recombinant tissue plasminogen activator (rt-PA). Use of selected ion extraction of the LC/MS data files allows one to graphically describe the elution order of closely related glycopeptides. The potential of LC/MS for the characterization of small amounts of unknown glycoproteins is shown by the study of an rt-PA mutant. A new potential site for glycosylation is created by site directed mutagenesis of wild type rt-PA with replacement of a threonine residue with asparagine at residue 103. An examination of a tryptic map shows that the mutant contains two new complex carbohydrate chains. The introduction of the new asparagine proximal to asparagine 117 changes this native high-mannose site in rt-PA to a complex-type glycosylation. This method allows rapid identification of carbohydrate containing peptides and yields useful structural information on microgram amounts of material.
Peptide mass mapping using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry in conjunction with interrogation of sequence databases is a powerful tool for the identification of proteins. Glycosylated proteins often yield poor MALDI peptide maps due to shielding of proteolytic cleavage sites and the presence of modified peptides. Here we demonstrate that enzymatic removal of N-linked glycans with simultaneous partial (50%) 18O-labeling of glycosylated asparagine residues prior to proteolysis and MALDI peptide mass mapping can overcome these problems. As a result, more peptides are observed in MALDI spectra which, in turn, increases the specificity of subsequent database searches. Furthermore, the detection of a labeled peptide directly translates into partial sequence information as N-linked carbohydrates are exclusively attached to asparagine residues that form part of the NXS/T sequence. The mass of the formerly glycosylated peptide together with the NXS/T sequence pattern represents a discriminating criterion for database searching which, on average, increases the search specificity by a factor of 100. This procedure allows the unambiguous identification of glycoproteins that would otherwise require sequencing and, at the same time, enables the identification of N-glycosylation sites with higher sensitivity than previously possible.
Analysis of the genome and proteome assumes the focus of attention in efforts to relate biochemical coding with cell functionality. Among other chores in energy metabolism, the talents of carbohydrates to establish a high-density coding system give reason for a paradigmatic shift. The sequence complexity of glycans and glycan-processing enzymes (glycosyltransferases, glycosidases and enzymes introducing substituents such as sulfotransferases), the growing evidence for the importance of glycans from transgenic and knock-out animal models and the correlation of defects in glycosylation with diseases are substantial assets to portray oligosaccharides as code words in their own right. Matching the pace of progress in the work on glycoconjugates, the increasing level of refinement of our knowledge about lectins (definition of this term: carbohydrate-binding proteins, excluding sugar-specific antibodies, receptors of free mono- or disaccharides for transport or chemotaxis and enzymes modifying the bound carbohydrate) epitomizes the sphere of action of the sugar code (functional lectinomics). It encompasses, among other activities, intra- and intercellular transport processes, sensor branches of innate immunity, regulation of cell-cell (matrix) adhesion or migration and positive/negative growth control with implications for differentiation and malignancy. The Q & A approach taken in this review lists a series of arguments in a stepwise manner to make the reader wonder why it is only a rather recent process that the concept of the sugar code has taken root in deciphering the mechanistic versatility of biological information storage and transfer.
The success attributed to identification and characterization of gel separated proteins by mass spectrometry (MS) is highly dependent on the percentage of an entire sequence covered by matching peptides derived from enzymatic digestion. Desalting and concentration of peptide mixtures on reversed-phase (RP) microcolumns prior to mass spectrometric analysis have resulted in increased signal-to-noise ratio and sensitivity, and consequently higher sequence coverage. A large proportion of peptides, however, remains undetected by MS presumably because they are lost during sample preparation on microcolumns, or are suppressed in the ionization process. We report here the use of graphite powder packed in constricted GELoader tips as an alternative to RP microcolumns for desalting and concentration of peptide mixtures prior to MS. Such columns are able to retain small and/or hydrophilic peptides that can be lost when using RP microcolumns. In addition, we show that samples contaminated with small biological polymers can readily be analyzed using graphite powder rather than RP microcolumns, since the polymer molecules bind strongly to graphite and are not eluted with the peptides.
Malignant transformation is associated with abnormal glycosylation, resulting in the synthesis and expression of altered carbohydrate determinants including sialyl Lewisa and sialyl Lewisx. The sialyl Lewisa and sialyl Lewisx determinants appear in the sera of patients with cancer, and are extensively utilized for serum diagnosis of cancers in Japan. Sialyl Lewisa and sialyl Lewisx are involved in selectin-mediated adhesion of cancer cells to vascular endothelium, and these determinants are thought to be closely associated with hematogenous metastasis of cancers. Recent progress in this area includes the following: 1. Substantial increases in solid clinical statistics that further confirm the contribution of these determinants in the progression of a wide variety of cancers; 2. Elucidation of the ligand specificity of the three family members of selectins and evaluation of the roles of these molecules in cancer cell adhesion; and 3. Advances in the study of the mechanism that leads to the enhanced expression of the sialyl Lewis(a/x) determinants in malignant cells. These recent results have confirmed that these determinants are not merely markers for cancers, but are functionally implicated in the malignant behavior of cancer cells. The results also suggested that the increase of these determinants in malignant cells is an inevitable consequence of the malignant transformation of cells. Considerable new knowledge has also been accumulated regarding the therapeutic implications for suppression of hematogenous metastasis targeting this cell adhesion system.
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.
This paper describes an efficient method of studying the glycoproteins found in snake venom. The glycosylation profiles of the Elapidae and Viperidae snake families were analyzed using FITC-labeled lectin glycoconjugates. The Con A-agarose affinity enrichment technique was used to fractionate glycoproteins from the N. naja kaouthia venom. The results revealed a large number of Con A binding glycoproteins, most of which have moderate to high molecular weights. To identify the proteins, the isolated glycoprotein fractions were subjected to two-dimensional electrophoresis and MALDI-TOF MS. Protein sequences were compared with published protein databases to determine for their biological functions.
The antiangiogenic activity of the multidomain plasma protein histidine-proline-rich glycoprotein (HPRG) is localized to its histidine-proline-rich (H/P) domain and has recently been shown to be mediated, at least partially, through binding to cell-surface tropomyosin in fibroblast growth factor-2-activated endothelial cells (X. Guan et al., Thromb Haemost, in press). HPRG and its H/P domain, but not the other domains of HPRG, bind specifically and with high affinity to tropomyosin. In this study, we characterize the interaction of the H/P domain with tropomyosin and delineate the region within the H/P domain responsible for that interaction. The H/P domain of HPRG consists mostly of repetitions of the consensus sequence [H/P][H/P]PHG. Applying an in vitro tropomyosin binding assay, we demonstrate that the synthetic peptide HHPHG binds to tropomyosin in vitro and inhibits angiogenesis and tumor growth in vivo. The affinity for tropomyosin increases exponentially upon multimerization of the HHPHG sequence, with a concurrent increase in antiangiogenic activity. Specifically, the tetramer (HHPHG)4 has significant antiangiogenic activity in the Matrigel plug model (IC50 approximately 600 nm) and antitumor effects in two syngeneic mouse tumor models. Thus, we show that a 16-mer peptide analogue mimics the antiangiogenic activity of intact HPRG and is also able to inhibit tumor growth, suggesting that cell surface tropomyosin may represent a novel antiangiogenic target for the treatment of cancer.
Glycosylation is one the most common post-translational modifications (PTM) and glycoproteins play fundamental roles in a diversity of biological processes. The development of an analytical approach to the study of variation of glycosylation patterns in serum samples has been hindered by the structural heterogeneity of this post-translational modification and the complexity of serum proteome. We have used the ability of different lectins to recognize specific glycosylation motifs to develop a specific affinity system that can achieve a comprehensive capture of serum glycoproteins. In a preliminary investigation, we evaluated the ability of five commonly used immobilized lectins to capture glycoproteins from human serum. SDS-PAGE analysis showed each lectin was able to enrich a subset of the serum glycoproteome and overlaps in lectin specificity were indeed observed. Based on these results and with the goal of studying the extent of the human serum glycoproteome, we then developed a multi-lectin affinity column containing Concanavalin A (Con A), Wheat germ and Jacalin lectin. The selection of lectins was also based on the known N-linked and O-linked glycan structures that are considered representative of the serum proteome. We then demonstrated that the capture of glycoproteins was specific, efficient and reproducible with this multi-lectin column. The results obtained with this affinity step indicated that about 10% of human serum proteins are glycosylated (weight/weight) and, after removal of six high abundance proteins, including albumin, at least 50% of the remaining proteins were glycosylated. We then evaluated the use of this affinity column to monitor changes in the pattern of glycosylation in serum samples by a controlled, stepwise release of the captured proteins from the multi-lectin affinity column with specific displacers.
It has been shown that oxidatively modified forms of proteins accumulate during oxidative stress, aging, and in some age-related diseases. One of the unique features of a wide variety of routes by which proteins are oxidized is the generation of carbonyl groups. This paper reports a method for the isolation of oxidized proteins, which involves (1) biotinylation of oxidized proteins with biotin hydrazide and (2) affinity enrichment using monomeric avidin affinity chromatography columns. The selectivity of the method was validated by adding in vitro oxidized biotinylated BSA to a yeast lysate and showing that the predominant protein recovered was BSA. This method was applied to the question of whether large doses of 2-nitropropane produce oxidized proteins. A study of rat liver homogenates showed that animals dosed with 2-nitropropane produced 17 times more oxidized protein than controls in 6 h. Tryptic digestion of these oxidized proteins followed by reversed-phase chromatography and tandem mass spectrometry led to the identification of 14 peptides and their parent proteins. Nine of the 14 identified peptides were found to carry 1 or 2 oxidation sites and 5 of the 9 peptides were biotinylated. The significance of this affinity method is that it allows the isolation of oxidized proteins from the rest of the proteome and facilitates their identification. In some cases, it is even possible to identify the site of oxidation.
This paper reports studies comparing the relative degree of sialylation among human serum glycoproteins carrying complex biantennary N-linked, hybrid, and high-mannose oligosaccharides. Comparisons were made by coupling lectin affinity selection with stable isotope coding of peptides from tryptic digests of serum. After proteolysis, samples were split and differentially acetylated with stable isotope coding agents according to either origin or the separation method by which they would be fractionated. A lectin column prepared from Sambucus nigra agglutinin (SNA) was used to select and compare the concentration of sialic acid containing glycopeptides. The relative standard deviation in quantification using this method was 4%. Using this method the concentration of sialic acid containing glycoproteins from a normal individual were compared to those in a pooled serum sample from a large number of normal individuals. It was found that sialylation varied less than 2-fold in all but four or five glycoproteins. Further studies were done on the degree of sialylation within glycoproteins. Samples labeled with the light isoform of the coding agent were applied to a set of serial lectin columns consisting of a concanavalin A (Con A) column coupled to an SNA column for selecting sialic acid appended to glycopeptides with complex biantennary N-linked, hybrid, and high-mannose glycans. In contrast, samples labeled with the heavy isoform of the coding agent were applied to a Con A lectin column alone to select glycopeptides containing complex biantennary N-linked, hybrid, and high-mannose glycans, without regard to sialylation. Glycopeptides thus selected were mixed, deglycosylated by PNGase F, and fractionated by reversed-phase chromatography (RPC). The RPC fractions were then analyzed by ESI-MS. The relative standard deviation of the method was 4%. All glycopeptides identified contained sialic acid except one. Peptides in which the relative abundance of isotopic isoforms was equal were considered to indicate that the protein parent was fully sialylated at that specific glycosylation site.
Serum profiling by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) holds promise as a clinical tool for early diagnosis of cancer and other human diseases. Sample preparation is key to achieving reproducible and well-resolved signals in MALDI-MS; a prerequisite for translation of MALDI-MS based diagnostic methods to clinical applications. We have investigated a number of MALDI matrices and several miniaturized solid-phase extraction (SPE) methods for serum protein concentration and desalting with the aim of generating reproducible, high-quality protein profiles by MALDI-MS. We developed a simple protocol for serum profiling that combines a matrix mixture of 2,5-dihydroxybenzoic acid and alpha-cyano-4-hydroxycinnamic acid with miniaturized SPE and MALDI-MS. Functionalized membrane discs with hydrophobic, ion-exchange or chelating properties allowed reproducible MALDI mass spectra (m/z 1000-12,000) to be obtained from serum. In a proof-of-principle application, SPE with chelating material and MALDI-MS identified protein peaks in serum that had been previously reported for distinguishing a person diagnosed with breast cancer from a control. These preliminary results indicate that this simple SPE/MALDI-MS method for serum profiling provides a versatile and scalable platform for clinical proteomics.
Biomarkers for cancer risk, early detection, prognosis, and therapeutic response promise to revolutionize cancer management. Protein biomarkers offer tremendous potential in this regard due to their great diversity and intimate involvement in physiology. An effective program to discover protein biomarkers using existing technology will require team science, an integrated informatics platform, identification and quantitation of candidate biomarkers in disease tissue, mouse models of disease, standardized reagents for analyzing candidate biomarkers in bodily fluids, and implementation of automation. Technology improvements for better fractionation of the proteome, selection of specific biomarkers from complex mixtures, and multiplexed assay of biomarkers would greatly enhance progress.
Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are posttranslationally processed proteins that become tethered to the extracellular leaflet of the plasma membrane via a C-terminal glycan-like moiety. Since the first GPI-AP was described in the 1970s, more than 500 GPI-APs have been reported in a range of species, including plants, microbes, and mammals. GPI-APs are probably involved in cell signaling, cell recognition, and cell remodeling processes, and they may potentially serve as cell surface antigens or vaccine targets in pathogenic microorganisms or transformed mammalian cells. Due to the structural complexity and physicochemical properties of GPI-APs, their identification and structural characterization is a demanding analytical task. Here, we report a simple, fast and sensitive method for isolation and structural analysis of GPI-anchors using a combination of hydrophilic interaction liquid chromatography and matrix-assisted laser desorption/ionization (MALDI) quadrupole time-of-flight tandem mass spectrometry. This method allowed analysis of GPI peptides derived from low picomole levels of the porcine kidney membrane dipeptidase. Furthermore, it allowed unambiguous assignment of the omega site via amino acid sequencing of the modified peptides. GPI-anchor-specific diagnostic ions were observed by MALDI-MS/MS at m/z 162, 286, 422, and 447, corresponding to glucosamine, mannose ethanolamine phosphate, glucosamine inositol phosphate, and mannose ethanolamine phosphate glucosamine, respectively. Thus, the methodology described herein may enable sensitive and specific detection of GPI-anchored peptides in large-scale proteomic studies of plasma membrane proteins.
In this paper, we describe the combination of lectin chromatography with capillary LC coupled to a linear ion trap-Fourier transform mass spectrometer (LTQ/FTMS) to enrich and characterize overexpressed glycoproteins from a cell culture lysate. A well-characterized glycoprotein, recombinant tissue plasminogen activator (rt-PA), was used as a standard, and we demonstrated that the three N-linked glycopeptides (including glycan structures) present in a tryptic digest of the rt-PA standard could be characterized in the new hybrid MS platform. A feature of this approach is that a significant amount of information can be obtained about the carbohydrate structures by direct analysis of the tryptic digest without the need for additional time-consuming sample preparation protocols. A combination of lectins was then studied for improved recovery of captured glycopeptides and was related to the selectivity of different lectins for specific glycosylation motifs. This approach was then extended to the lysate of a cell line routinely used in biotechnology manufacture (Chinese hamster ovary, CHO). This study showed that the combinations of lectins could enrich glycoproteins significantly from a CHO cell lysate. We also demonstrated that with this level of enrichment and with the new hybrid mass spectrometer, we could study the structures of N-linked glycopeptides of rt-PA present in a crude CHO cell lysate, at a ratio of 1:200 (rtPA:total cell lysate protein, w/w) by accurate mass measurement in the FTMS and tandem MSn in the linear ion trap. The generic and high throughput nature of the lectin approach combined with the ability to directly analyze the glycan structures in the tryptic digest suggest that this platform has the potential to routinely monitor glycoprotein products at early stage manufacturing in the biotech industry.
Crystal structures of the lectin and epidermal growth factor (EGF)-like domains of P-selectin show 'bent' and 'extended' conformations. An extended conformation would be 'favored' by forces exerted on a selectin bound at one end to a ligand and at the other end to a cell experiencing hydrodynamic drag forces. To determine whether the extended conformation has higher affinity for ligand, we introduced an N-glycosylation site to 'wedge open' the interface between the lectin and EGF-like domains of P-selectin. This alteration increased the affinity of P-selectin for its ligand P-selectin glycoprotein 1 (PSGL-1) and thereby the strength of P-selectin-mediated rolling adhesion. Similarly, an asparagine-to-glycine substitution in the lectin-EGF-like domain interface of L-selectin enhanced rolling adhesion under shear flow. Our results demonstrate that force, by 'favoring' an extended selectin conformation, can strengthen selectin-ligand bonds.
Glycoproteins are often associated with cancer and are important in serum studies, for which glycosylation is a common posttranslational modification.
We used multilectin affinity chromatography (M-LAC) to isolate glycoproteins from the sera of breast cancer patients and controls. The proteins were identified by HPLC-tandem mass spectrometry (MS/MS) analysis of the corresponding tryptic digests. We used the FuncAssociate Gene Ontology program for association analysis of the identified proteins. Biomarker candidates in these groups were comparatively quantitated by use of peak area measurements, with inclusion of an internal standard. We analyzed data for concordance within the ontology association groups for vector of change with the development of breast cancer.
Detection of the known low-concentration biomarker HER-2 (8-24 mug/L) enabled us to establish a dynamic range of 10(6), relative to the amount of albumin, for the depletion step. We then used ELISA to confirm this range. Proteins associated with lipid transport and metabolism, cell growth and maintenance, ion homeostasis, and protease inhibition were found to be differentially regulated in serum from women with breast cancer compared with serum from women without breast cancer.
M-LAC for isolation of the serum glycoproteome, coupled with liquid chromatography-MS/MS and the use of gene ontology associations, can be used to characterize large panels of candidate markers, which can then be evaluated in a particular patient population.
Separation of polar compounds on polar stationary phases with partly aqueous eluents is by no means a new separation mode in LC. The first HPLC applications were published more than 30 years ago, and were for a long time mostly confined to carbohydrate analysis. In the early 1990s new phases started to emerge, and the practice was given a name, hydrophilic interaction chromatography (HILIC). Although the use of this separation mode has been relatively limited, we have seen a sudden increase in popularity over the last few years, promoted by the need to analyze polar compounds in increasingly complex mixtures. Another reason for the increase in popularity is the widespread use of MS coupled to LC. The partly aqueous eluents high in ACN with a limited need of adding salt is almost ideal for ESI. The applications now encompass most categories of polar compounds, charged as well as uncharged, although HILIC is particularly well suited for solutes lacking charge where coulombic interactions cannot be used to mediate retention. The review attempts to summarize the ongoing discussion on the separation mechanism and gives an overview of the stationary phases used and the applications addressed with this separation mode in LC.