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Synergic effect of multiple functional groups for glycopeptide enrichment (a) Mercaptobenzene boronic acids grated Au-doped straticulate C 3 N 4 ; (b) Ti 4þ -immobilized dendritic polyglycerol CS@PGMA@IDA (CS, chitosan; PGMA, poly(glycidyl methacrylate); IDA, iminodiacetic acid) material for glycopeptide and phosphopeptide simultaneous enrichment. Adapted with permission [85], Copyright 2018, Spinge. Adapted with permission [87], Copyright 2016, the Royal Society of Chemistry.
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Glycosylation is one of the most common and important types of protein post-translational modifications in mammalian cells. To decode the protein glycosylation, it is highly important to enrich glycopeptides or separate glycans from complex biosamples. Hydrophilic interaction liquid interaction chromatography (HILIC) has inherent advantages in the...
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Context 1
... group, integration of two or three types of functional groups into one system has been reported and notably improved the interaction between adsorbent and glycopeptide [83,84]. Recently, Zhang et al. grafted mercaptobenzene boronic acids onto surface of Au-doped straticulate C 3 N 4 (designated as MASC) via a facile and economical procedure (Fig. 8a). The resulting MASC material demonstrated a satisfactory adaptation to low pH value and excellent hydrophilicity. A total of 1465 N-linked glycopeptides from 839 glycoproteins and 463 O-linked glycopeptides from 209 glycoproteins were selectively captured from human urine and serum, respectively. High enrichment efficiency of MASC was ...
Context 2
... and glycopeptides [85]. In 2016, Zou et al. coated iminodiacetic acid on chitosan@poly(glycidyl methacrylate) (denoted as CS@PGMA@IDA) nanomaterials and employed CS@PGMA@IDA to selectively bind N-linked glycopeptides [86]. Recently, they immobilized Ti 4þ on the surface CS@PGMA@IDA and obtained nanomaterials with both HILIC and IMAC properties (Fig. 8b). CS@PGMA@IDA-Ti 4þ could enrich low-abundance Nlinked glycopeptides and phosphopeptides simultaneously, which was simple, efficient, and selective [87]. Zhang's group successfully prepared a 3D-branched copolymer modified silica via a thiol-ene click copolymerization. The material featured with hydrophilic interaction and ...
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Citations
... Nevertheless, the HILIC strategy is not without its limitations, particularly in terms of achieving highly selective enrichment. For instance, some HILIC materials are capable of enriching neutral or positively charged glycopeptides, but are less effective at enriching negatively charged glycopeptides [56]. Additionally, the introduction of positively charged metal ions can enhance the binding ability of HILIC materials to negatively charged glycopeptides. ...
Endogenous peptides, particularly those with post-translational modifications, are increasingly being studied as biomarkers for diagnosing various diseases. However, they are weakly ionizable, have a low abundance in biological samples, and may be interfered with by high levels of proteins, peptides, and other macromolecular impurities, resulting in a high limit of detection and insufficient amounts of post-translationally modified peptides in real biological samples to be examined. Therefore, separation and enrichment are necessary before analyzing these biomarkers using mass spectrometry. Mesoporous materials have regular adjustable pores that can eliminate large proteins and impurities, and their large specific surface area can bind more target peptides, but this may result in the partial loss or destruction of target peptides during centrifugal separation. On the other hand, magnetic mesoporous materials can be used to separate the target using an external magnetic field, which improves the separation efficiency and yield. Core–shell magnetic mesoporous materials are widely utilized for peptide separation and enrichment due to their biocompatibility, efficient enrichment capability, and excellent recoverability. This paper provides a review of the latest progress in core–shell magnetic mesoporous materials for enriching glycopeptides and phosphopeptides and compares their enrichment performance with different types of functionalization methods.
... HILIC was chosen to achieve the separation of surrogate glycopeptides due to its retention of hydrophilic substances. 58 NIST mAb IgG was first trypsin digested. Five tyrosinecontaining glycopeptides having a glycan at the N297 Fc residue of the heavy chain, EEQYNSTYR-G0F, EEQYNSTYR-G1F, EEQYNSTYR-G2F, EEQYNSTYR-G1F(-GlcNAc), and EE-QYNSTYR-G2F(Hex) (listed in Table 1), where the glycans attached in the residue of asparagine (N, see the glycopeptide structures in Table 1 and Figure 3) were separated out by a HILIC column with retention times at 25.5, 27.4, ...
... Studies have shown that MOFs can interact with proteins through various mechanisms, including electrostatic interactions, hydrogen bonding, and hydrophobic interactions. 75 These interactions can affect the conformation and stability of proteins, which may have implications for their biological function. In addition to proteins, MOFs can also interact with cells. ...
Metal-organic frameworks (MOFs) have garnered significant attention as versatile materials with diverse applications in various scientific domains. Their unique structural characteristics and tunable properties make them promising candidates for biomedical applications such as drug delivery, imaging, and tissue engineering. However, the cytotoxicity and biocompatibility of MOFs are critical considerations that must be thoroughly evaluated to ensure their safe and effective use in biological systems. The impact of MOF physicochemical properties on cellular uptake mechanisms and intracellular fate is examined, shedding light on the underlying mechanisms of MOF-cell interactions. Moving beyond in vitro evaluations, the chapter delves into in vivo studies that elucidate the biodistribution, clearance, and long-term effects of MOFs in living organisms. Furthermore, the chapter examines strategies for enhancing the biocompatibility of MOFs through surface modifications, encapsulation, and co-delivery approaches. The integration of computational modeling in predicting MOF behavior within biological systems is also discussed, offering insights into rational design principles for minimizing cytotoxic effects. In conclusion, this chapter underscores the significance of understanding the cytotoxicity and biocompatibility of MOFs in the context of their biomedical applications. As MOFs continue to advance the frontier of nanomedicine, their safety profile remains paramount. A comprehensive evaluation of MOF-cell interactions, combined with innovative design strategies, will pave the way for the development of MOF-based therapeutics and diagnostics that offer efficacious and biocompatible solutions to pressing medical challenges.
... One of the critical issues is the relatively weak hydrophilic affinity of adsorbents, which leads to the loss of glycopeptides during the enrichment process. 22,23 Previously, we have demonstrated that increasing the density of hydrophilic groups on the absorbent surface can effectively enhance glycopeptide affinity. 24 Nevertheless, the actual HILIC mechanism is considerably intricate, involving not only partition-driven phenomena but also adsorption, including hydrogen bonding, dipole−dipole interactions, and attractive or repulsive electrostatic interactions. ...
... 25 According to the "hydrophilic subtraction" model, hydrogen bonding interaction primarily accounts for the retention mechanism in neutral functional group-based HILIC (e.g., carbohydrate, amide, and diol) while electrostatic interactions predominantly contribute to zwitterion-based HILIC. 22 Kawachi et al. investigated 14 kinds of HILIC stationary phases and classified them into two clusters: strongly hydrophilic phases, including amide and zwitterion, and weakly hydrophilic phases, such as cyclodextrin, diol, triazole, and amine. 26 These results suggest that novel HILIC materials should be tailored based on the chemical properties of various hydrophilic groups to manipulate glycopeptide selectivity and retention. ...
Protein glycosylation is ubiquitous and crucial for regulating biological processes in organisms. Given the hetero-geneity and low abundance of glycoproteins, efficient and specific enrichment procedures are required for the mass spectrometry analysis of glycopeptides. Hydrophilic interaction liquid chromatography (HILIC) has emerged as an effective strategy for glycopeptide enrichment. However, the relatively weak hydrophilic affinity restricts the achievement of a satisfactory enrichment performance. Here, we presented a rational design of an amide and multihydroxyl complementary tailored metal−organic framework, denoted as U6N/Pv@Glc, which exhibited ultrahydrophilicity and enhanced glycan affinity. Our results demonstrated a significant increase in glycopeptide coverage after enrichment, accompanied by extremely low detection limits (0.05 fmol μL −1) and high selectivity (IgG/BSA, 1:4000) as evaluated using trypsin-digested standard glycoproteins. A total of 379 glycopeptides and 247 intact glycopeptides (containing a total of 1577 site-specific N-glycans) were identified and characterized within human serum samples from individuals with type 2 diabetes in-depth. Additionally, we extended the application of this material to capture undigested glycoproteins, demonstrating potential compatibility with top-down MS analysis. These results highlight the promising potential of this novel material for comprehensive glycoproteomic analysis of every potential aspect.
... Kromatografi uygulamaları, glikanları sahip oldukları, boyut, yük ve hidrofobiklik gibi fizikokimyasal özelliklerine ayırabildiğinden, glikan ayrımı için yaygın olarak kullanılmaktadır [32]. Literatürde, glikanların ayrımı için kullanılmış farklı kromatografi çeşitleri bulunmaktadır. ...
... İyon Değiştirme Kromatografisi (IEC) [27][28][29][30][31][32][33][34][35][36] İyon değiştirici gruplar içeren dolgu maddeleri Glikanların yüklü bölgelerine göre ayrılması. ...
Glikanlar, çeşitli biyolojik süreçlerde önemli rol oynayan ve sağlık ile hastalık üzerinde önemli etkileri olan karmaşık karbonhidrat molekülleri olarak bilinmektedir. Glikanların kapsamlı bir şekilde analiz edilmesi, gelişmiş analitik tekniklerin bir kombinasyonunu gerektirmektedir. Bu derleme, glikan analizinde kullanılan çeşitli tekniklerin, örnekleme hazırlığı, glikan zenginleştirme, glikan salımı, etiketleme, ayrıştırma ve tespit gibi adımlarının ayrıntılı bir iş akışını sunmaktadır. Her adımın prensipleri, uygulamaları ve avantajları açıklanarak, glikan araştırmalarına katkıları vurgulanmaktadır. Ayrıca, spesifik glikan analiz hedefleri için uygun tekniklerin seçiminin önemi üzerinde durulmaktadır. Bu iş akışı, glikanların kapsamlı bir anlayışını sağlayarak, biyolojik sistemlerdeki rollerini açığa çıkarmaya ve yeni terapötik müdahalelerin geliştirilmesine yardımcı olmaktadır.
... Recently, accumulating evidences have shown that exosome cargos can be transferred from donor cells to recipient cells, and therefore, they can reflect the altered physiological and pathological state of the corresponding donor cells [6][7][8][9][10]. As the most abundant post-translational modification (PTM) process, glycosylation has shown great effort in exosome biology [11][12][13][14]. And the aberrant alteration of exosomal glycans has attracted more and more attention for disease monitoring [15][16][17]. ...
Soft-template carbonized mesopores were developed for the purpose of enriching urinary exosomal glycans through organic–organic self-assembly using block copolymers and resol precursors. With a high surface area of 229 m² g⁻¹, a small pore size of 3.1 nm, and a significant amount of carbon that specifically interacts with oligosaccharides in glycans, this carbonized mesopore material exhibits high selectivity and low limits of detection (5 ng μL⁻¹) towards glycans. Our analysis of complex urine samples from healthy volunteers and bladder carcinoma patients successfully profiled 48 and 56 exosomal glycans, respectively, and 16 of them were significantly changed. Moreover, one upregulated bisecting N-acetylglucosamine (GlcNAc)-type glycan with core fucose, two upregulated and two downregulated terminal-sialylated glycans were revealed to be linked to bladder carcinoma. This approach is of significant importance for understanding diseases that arise from protein glycosylation mutations, and it may contribute to the development of novel diagnostic and therapeutic strategies for bladder carcinoma.
Graphical Abstract
... For mass spectrometric (MS) analysis, it is crucial to enrich glycopeptides from complex biological samples due to their low abundance, presence of interfering species, and low ionization efficiency. Strategies have been developed for specific enrichment of glycopeptides [8] which include hydrazide chemistry [9], boronic acid affinity chromatography [10], lectin-affinity chromatography [11] and hydrophilic interaction chromatography (HILIC) [12]. HILIC has high selectivity, reproducibility, ease of operation and unbiased enrichment of glycopeptides. ...
HILIC (hydrophilic interaction liquid chromatography) materials enrich glycopeptides. The non-specific interactions because of support material and inadequate hydrophilicity render loss of less abundant glycopeptides in SPE-based enrichments. In this work, magnetic terpolymer (Fe3O4@MAA/DVB/1,2-Epoxy-5-hexene) is functionalized with Ranachrome-5 to generate enhanced hydrophilicity. Amine, carboxylic, and amide groups of ranachrome-5 provide zwitterionic chemistry. Material's magnetic core contributes to ease of operation while higher surface area 97.0711 m2 g-1 immobilizes better quantities of Ranachrome-5. Homogeneous morphology, nano-size, and super hydrophilicity enhance enrichment. Ranachrome-5 functionalized polymeric core-shell beads enrich 25, 18 and 16 N-linked glycopeptides via SPE strategy from tryptic digests of model glycoproteins i.e., immunoglobulin G (IgG), horseradish peroxidase (HRP) and chicken avidin, respectively. Zwitterionic chemistry of ranachrome-5 helps in achieving higher selectivity (1:250, HRP / Bovine Serum Albumin), and lower detection limit (100 attomole, HRP digest) with complete glycosylation profile of each standard digest. High binding capacity (137.1 mg/g) and reuse of affinity material up to seven cycles reduce the cost and amount of affinity material for complex sample analysis. A recovery of 91.76% and relative standard deviation (RSD) values less than 1 define the application of HILIC beads for complex samples like plasma. 508 N-linked intact low abundant glycopeptides corresponding to 50 glycoproteins are identified from depleted human plasma samples via nano-Liquid Chromatography-Tandem Mass Spectrometry (nLC-MS/MS). Using Single Nucleotide Variances (BioMuta) for low abundant plasma glycoproteins, the potential association of proteins to four cancers, i.e., breast, lung, uterine, and melanoma is evaluated. Via the bottom-up approach, HILIC beads can analyze clinically important low-abundant glycoproteins.
... To solve this problem, hydrophilic interaction liquid chromatography (HILIC) was first reported in 1990 by Alpert et al. [1]. Unlike NPLC, HILIC can achieve complete separation of polar compounds with a hydrophilic stationary phase and high concentrations of organic solvents, effectively solving the problems of solubility and compatibility with mass spectrometry [2][3][4][5][6]. HILIC has therefore become a good alternative with high selectivity for polar analytes in many fields, including carbohydrates [7][8][9], glycopeptides [10,11], peptides [12,13], amino acids [7,14,15], metabolites [16], small drug molecules [17,18], and polar natural products [19,20]. ...
Two novel stationary phases, 1-(4-bromobutyl)-3-methylimidazolium bromide bonded chitosan modified silica and 1-(4-bromobutyl)-3-methylimidazolium bromide bonded chitosan derivatized calix[4]arene modified silica stationary phase, were synthesized using 1-(4-bromobutyl)-3-methylimidazolium bromide bonding chitosan as a polarity regulator solving the limitation of the strong hydrophobicity of calixarene in the application of hydrophilic field. The resulting materials were characterized by solid-state nuclear magnetic resonance, Fourier-transform infrared spectra, scanning electron microscopy, elemental analysis, and thermogravimetric analysis. Based on the hydrophilicity endowed by 1-(4-bromobutyl)-3-methylimidazolium bromide bonded chitosan, the retention mode of ILC-Sil and ILCC4-Sil could be effectively switched from the hydrophilic mode to a hydrophilic/hydrophobic mixed mode and could simultaneously provide various interactions with solutes, including hydrophilic, π-π, ion-exchange, inclusion, hydrophobic, and electrostatic interactions. On the basis of these interactions, successful separation and higher shape selectivity were achieved among compounds that vary in polarity under both reverse-phase and hydrophilic interactive liquid chromatography conditions. Moreover, the ILCC4-Sil was successfully applied to the determination of morphine in actual samples using solid-phase extraction and mass spectrometry. The LOD and LOQ were 15 pg/mL and 54 pg/mL, respectively. This work presents an exceptionally flexible adjustment strategy for the retention and selectivity of a silica stationary phase by tuning the modification group.
Graphical Abstract
... This fact makes it suitable for the analysis of free urinary oligosaccharides. For a detailed description of HILIC advantages, we refer to two comprehensive reviews published on this topic [21,22]. ...
Background:
Alpha-mannosidosis is a rare lysosomal storage disorder, caused by decreased activity of α-D-mannosidase. This enzyme is involved in the hydrolysis of mannosidic linkages in N-linked oligosaccharides. Due to the mannosidase defect, undigested mannose-rich oligosaccharides (Man2GlcNAc - Man9GlcNAc) accumulating in cells are excreted in large quantities in urine.
Methods:
In this work, we determined the levels of urinary mannose-rich oligosaccharides in a patient subjected to novel enzyme replacement therapy. Urinary oligosaccharides were extracted using solid phase extraction (SPE), labeled by fluorescent tag 2-aminobenzamide, and quantified by high-performance liquid chromatography (HPLC) with fluorescence detector (FLD). The identity of peaks was determined by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry. In addition, the levels of urinary mannose-rich oligosaccharides were also quantified by 1H nuclear magnetic resonance (NMR) spectroscopy. The data were analyzed using one-tailed paired t-test and Pearson's correlation tests.
Results:
Compared to levels before the administration of therapy, an approximately two-folds decrease in total mannose-rich oligosaccharides after one month of treatment was observed by NMR and HPLC. After four months, an approximately ten-folds significant decrease in total urinary mannose-rich oligosaccharides was detected, suggesting therapy effectiveness. A significant decrease in the levels of oligosaccharides with 7-9 mannose units was detected by HPLC.
Conclusions:
The application of both HPLC-FLD and NMR in quantification of oligosaccharide biomarkers is a suitable approach for monitoring of therapy efficacy in alpha-mannosidosis patients.
... A polar functional group is needed for HILIC, and it can be neutral or charged, such as sugars, amino acids, amides, amines, metal-organic frameworks (MOFs), and covalent organic frameworks (COFs). All these materials and their applications for glycopeptide analysis have been recently summarized elsewhere [11]. In HILIC applications, the use of hydrogels for preparation of new materials has been recently suggested for N-glycopeptide sample preparation. ...
This trends article provides an overview of the state of the art in the analysis of intact glycopeptides by proteomics technologies based on LC–MS analysis. A brief description of the main techniques used at the different steps of the analytical workflow is provided, giving special attention to the most recent developments. The topics discussed include the need for dedicated sample preparation for intact glycopeptide purification from complex biological matrices. This section covers the common approaches with a special description of new materials and innovative reversible chemical derivatization strategies, specifically devised for intact glycopeptide analysis or dual enrichment of glycosylation and other post-translational modifications. The approaches are described for the characterization of intact glycopeptide structures by LC–MS and data analysis by bioinformatics for spectra annotation. The last section covers the open challenges in the field of intact glycopeptide analysis. These challenges include the need of a detailed description of the glycopeptide isomerism, the issues with quantitative analysis, and the lack of analytical methods for the large-scale characterization of glycosylation types that remain poorly characterized, such as C-mannosylation and tyrosine O-glycosylation. This bird’s-eye view article provides both a state of the art in the field of intact glycopeptide analysis and open challenges to prompt future research on the topic.
Graphical Abstract
