Enrichment Of Glycopeptides Research Articles

Post-synthesis modification of covalent organic frameworks for ultrahigh enrichment of low-abundance glycopeptides from human saliva and serum

In this study, a novel type of covalent organic framework (COF) material rich in boronic acid sites was prepared through post-synthesis modification (TbBD@PEI@Au@4-MPBA). The surface of COF material had abundant carboxylic acid groups, which could bind a large amount of polyethyleneimine (PEI) through electrostatic interaction. At the same time, the amino groups on the PEI can be grafted with Au nanoparticles (Au NPs) in situ, and then 4-mercaptophenylboronic acid (4-MPBA) was modified by the reaction of Au and sulfhydryl groups. The massive grafting of boronic acid groups made the material's enrichment effect on glycopeptides expected. The results of experiments indicated that the composite material has high sensitivity (5 amol μL−1) and selectivity (1:1000). In addition, the material has outstanding stability and reusability, with a load capacity of about 100 mg g−1 and a recovery of 99.3 ± 2.2%. What's more, after enriched by TbBD@PEI@Au@4-MPBA, 56 endogenous glycopeptides from fresh human saliva were detected by MALDI-TOF MS, 56 unique glycopeptides corresponding to 31 glycoproteins from human saliva and 513 unique glycopeptides corresponding to 208 glycoproteins from serum of throat cancer patient were detected by nano-LC-MS/MS, respectively, which was expected to be applied to glycoproteomics research.

ZIC-cHILIC Functionalized Magnetic Nanoparticle for Rapid and Sensitive Glycopeptide Enrichment from

Due to their unique glycan composition and linkage, protein glycosylation plays significant roles in cellular function and is associated with various diseases. For comprehensive characterization of their extreme structural complexity occurring in >50% of human proteins, time-consuming multi-step enrichment of glycopeptides is required. Here we report zwitterionic n-dodecylphosphocholine-functionalized magnetic nanoparticles (ZIC-cHILIC@MNPs) as a highly efficient affinity nanoprobe for large-scale enrichment of glycopeptides. We demonstrate that ZIC-cHILIC@MNPs possess excellent affinity, with 80–91% specificity for glycopeptide enrichment, especially for sialylated glycopeptide (90%) from biofluid specimens. This strategy provides rapidity (~10 min) and high sensitivity (<1 μL serum) for the whole enrichment process in patient serum, likely due to the rapid separation using magnetic nanoparticles, fast reaction, and high performance of the affinity nanoprobe at nanoscale. Using this strategy, we achieved personalized profiles of patients with hepatitis B virus (HBV, n = 3) and hepatocellular carcinoma (HCC, n = 3) at the depth of >3000 glycopeptides, especially for the large-scale identification of under-explored sialylated glycopeptides. The glycoproteomics atlas also revealed the differential pattern of sialylated glycopeptides between HBV and HCC groups. The ZIC-cHILIC@MNPs could be a generic tool for advancing the glycoproteome analysis, and contribute to the screening of glycoprotein biomarkers.

Systems-wide analysis of glycoprotein conformational changes by limited deglycosylation assay

A new method to probe the conformational changes of glycoproteins on a systems-wide scale, termed limited deglycosylation assay (LDA), is described. The method measures the differential rate of deglycosylation of N-glycans on natively folded proteins by the common peptide:N-glycosidase F (PNGase F) enzyme which in turn informs on their spatial presentation and solvent exposure on the protein surface hence ultimately the glycoprotein conformation. LDA involves 1) protein-level N-deglycosylation under native conditions, 2) trypsin digestion, 3) glycopeptide enrichment, 4) peptide-level N-deglycosylation and 5) quantitative MS-based analysis of formerly N-glycosylated peptides (FNGPs). LDA was initially developed and the experimental conditions optimized using bovine RNase B and fetuin. The method was then applied to glycoprotein extracts from LLC-MK2 epithelial cells upon treatment with dithiothreitol to induce endoplasmic reticulum stress and promote protein misfolding. Data from the LDA and 3D structure analysis showed that glycoproteins predominantly undergo structural changes in loops/turns upon ER stress as exemplified with detailed analysis of ephrin-A5, GALNT10, PVR and BCAM. These results show that LDA accurately reports on systems-wide conformational changes of glycoproteins induced under controlled treatment regimes. Thus, LDA opens avenues to study glycoprotein structural changes in a range of other physiological and pathophysiological conditions relevant to acute and chronic diseases. SignificanceWe describe a novel method termed limited deglycosylation assay (LDA), to probe conformational changes of glycoproteins on a systems-wide scale. This method improves the current toolbox of structural proteomics by combining site and conformational-specific PNGase F enzymatic activity with large scale quantitative proteomics. X-ray crystallography, nuclear magnetic resonance spectroscopy and cryoEM techniques are the major techniques applied to elucidate macromolecule structures. However, the size and heterogeneity of the oligosaccharide chains poses several challenges to the applications of these techniques to glycoproteins. The LDA method presented here, can be applied to a range of pathophysiological conditions and expanded to investigate PTMs-mediated structural changes in complex proteomes.

One-Step Synthesis of Hydrophilic Nano-Floral Inter-Polymeric Material for Highly Selective Enrichment of N-Linked Glycopeptides

A novel nano-floral inter-polymeric material (λ-Carr-Ag-SA) was developed to effectively capture N-linked glycopeptides from biological samples. A simple one-step reduction of silver ions was involved in the synthesis of the material in which λ-carrageenan and sodium alginate, with abundant hydrophilic groups, were used as the reducing agent and stabilizer. The performance of λ-Carr-Ag-SA was evaluated experimentally by enriching glycopeptides from horse radish peroxidase (HRP) and human serum. All the results demonstrated that λ-Carr-Ag-SA had a great selectivity, low detection limit and a large binding capacity toward glycopeptides. The successful application exhibits great potential of λ-Carr-Ag-SA for further glycoproteome analysis.

Fabrication of magnetic dual-hydrophilic metal organic framework for highly efficient glycopeptide enrichment.

Highly selective glycopeptide enrichment is important before mass spectrometry analysis because of the ultra-low abundance of glycopeptides in the peptide mixtures. Herein, a UiO-66-NH2-based magnetic composite was prepared and used for the hydrophilic enrichment of glycopeptides. The composite was modified with phytic acid (PA) molecules by partially replacing 2-aminoterephthalic acid ligands in UiO-66-NH2, with electrostatic interactions also promoting this modification process. Based on the hydrophilicity of both the PA molecules and the UiO-66-NH2 skeleton, the resulting material, denoted as MUiO-66-NH2/PA, showed excellent dual hydrophilicity towards glycopeptide enrichment. Compared with pure UiO-66-NH2, the specific surface area and hydrophilicity of the prepared material were increased, and MUiO-66-NH2/PA exhibited good magnetic responsiveness to facilitate a convenient enrichment procedure. HRP and IgG were used as standard proteins to evaluate the glycopeptide enrichment properties, with 21 and 34 glycopeptides enriched from their tryptic digests. Furthermore, MUiO-66-NH2/PA showed outstanding sensitivity (1 fmol/μL) and selectivity (HRP/BSA = 1:1000), and achieved remarkable glycopeptide enrichment performance for practical human serum samples. Notably, MUiO-66-NH2/PA showed perfect reusability and stability, achieving enrichment performance after five cycles similar to that of the first use. This material can be used for glycopeptide enrichment to obtain further glycosylation information, providing the possibility for cancer treatment.

Glutathione-functionalized two-dimensional cobalt sulfide nanosheets for rapid and highly efficient enrichment of N-glycopeptides.

Protein glycosylation plays pivotal role in a variety of biological processes and has association with many diseases. The highly efficient glycopeptide enrichment is essential for the mass spectrometry-based glycoproteome research to reduce interference from non-glycopeptides. In this study, novel glutathione-functionalized two-dimensional cobalt sulfide nanosheets (Co-S@Au-GSH) were synthesized for rapid and highly effective enrichment of glycopeptides. By using this nanomaterial, 34 and 21 N-glycopeptides were effectively captured from human serum immunoglobulin G (IgG) and horseradish peroxidase (HRP) digests, respectively. In addition, the Co-S@Au-GSH showed remarkable performance in N-glycopeptide extraction with high selectivity (HRP: BSA = 1:500), low limit of detection (0.5 fmol/μL), high binding capacity (150 mg/g), good reusability, and great robustness. Moreover, it was successfully applied in complex serum samples, demonstrating its excellent enrichment performance. These results indicated that this nanomaterial has great potential in complicated practice samples in glycoproteome determination.

Comprehensive O-Glycosylation Analysis of the SARS-CoV-2 Spike Protein with Biomimetic Trp-Arg Materials.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a serious public health threat. Most vaccines against SARS-CoV-2 target the highly glycosylated spike protein (S). A good knowledge of the glycosylation profile of this protein is key to successful vaccine development. Unlike the 22 confirmed N-glycosylation sites on SARS-CoV-2 S, only a few O-glycosylation sites on this protein have been reported. This difference is mainly ascribed to the extremely low stoichiometry of O-glycosylation. Herein, we designed the biomimetic materials, Trp-Arg (WR) monomer-grafted silica microspheres (designated as WR-SiO2), and these biomimetic materials can enrich N- and O-linked glycopeptides with high selectivity. And WR-SiO2 can resist the nonglycopeptides' interference with the 100 molar fold of BSA during O-linked glycopeptide enrichment. We utilized WR-SiO2 to comprehensively analyze the O-glycosylation profile of recombinant SARS-CoV-2 S. Twenty-seven O-glycosylation sites including 18 unambiguous sites are identified on SARS-CoV-2 S. Our study demonstrates that the biomimetic polymer can offer specific selectivity for O-linked glycopeptides and pave the way for O-glycosylation research in biological fields. The O-glycosylation profile of SARS-CoV-2 S might supplement the comprehensive glycosylation in addition to N-glycosylation of SARS-CoV-2 S.

Glycosylation States on Intact Proteins Determined by NMR Spectroscopy.

Protein glycosylation is important in many organisms for proper protein folding, signaling, cell adhesion, protein-protein interactions, and immune responses. Thus, effectively determining the extent of glycosylation in glycoprotein therapeutics is crucial. Up to now, characterizing protein glycosylation has been carried out mostly by liquid chromatography mass spectrometry (LC-MS), which requires careful sample processing, e.g., glycan removal or protein digestion and glycopeptide enrichment. Herein, we introduce an NMR-based method to better characterize intact glycoproteins in natural abundance. This non-destructive method relies on exploiting differences in nuclear relaxation to suppress the NMR signals of the protein while maintaining glycan signals. Using RNase B Man5 and RNase B Man9, we establish reference spectra that can be used to determine the different glycoforms present in heterogeneously glycosylated commercial RNase B.

One-step fabrication of strongly hydrophilic mesoporous silica for comprehensive analysis of serum glycopeptidome

Glycopeptidome represents reliable predictors of physiological and pathological status. Obstructions mainly including low abundance of endogenous glycopeptides and varied interference necessitate glycopeptide enrichment prior to MS analysis. Inspired by the prevalence of hydrophilic interaction chromatography for glycopeptide enrichment, a novel magnetic mesoporous silica nanomaterial (Fe3O4@mSiO2-TSG) with strongly hydrophilic property was developed through a one-pot method. In this work, the gluconamide-containing organosilane is innovatively proposed to directly serve as the strongly hydrophilic silica source for fabrication of hydrophilic mesoporous silica nanomaterial for glycopeptidomics research. Apart from excellent hydrophilicity, Fe3O4@mSiO2-TSG also was equipped with large specific surface area, ordered mesopore channels and great magnetic responsiveness. With all the advantages, Fe3O4@mSiO2-TSG displayed remarkable size-exclusion effect and considerable reusability. Moreover, combined with nano-LC-MS/MS, the glycopeptidome of serum from breast cancer patients was analyzed comprehensively, which showed noteworthy difference from healthy serum through gene ontology analysis, indicating great potential of the approach for glycopeptidomics research.

High-Throughput Analyses of Glycans, Glycosites, and Intact Glycopeptides Using C4-and C18/MAX-Tips and Liquid Handling System.

Protein glycosylation is one of the most common and diverse modifications. Aberrant protein glycosylation has been reported to associate with various diseases. High‐throughput and comprehensive characterization of glycoproteins is crucial for structural and functional studies of altered glycosylation in biological, physiological, and pathological processes. In this protocol, we detail a workflow for comprehensive analyses of intact glycopeptides (IGPs), glycosylation sites, and glycans from N‐linked glycoproteins. By utilizing liquid handling systems, our workflow could enrich IGPs in a high‐throughput manner while reducing sample processing time and human error involved in traditional proteomics sample processing techniques. Together, our workflow enables a high‐throughput enrichment of glycans, glycosites, and intact glycopeptides from complex biological or clinical samples. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Enzymatic digestion of glycoproteins using C4‐tipsBasic Protocol 2: Intact glycopeptide analysis using C18/MAX‐tipsBasic Protocol 3: Glycan and glycosite analysis

Simultaneous enrichment and separation of neutral and sialyl glycopeptides of SARS-CoV-2 spike protein enabled by dual-functionalized Ti-IMAC material.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a serious threat to human health all over the world. The development of effective vaccines has been focusing on the spike (S) glycoprotein, which mediates viral invasion to human cells through its interaction with the angiotensin-converting enzyme 2 (ACE2) receptor. In this work, we perform analytical characterization of N- and O-linked glycosylation of the SARS-CoV-2 S glycoprotein. We explore the novel use of dual-functionalized titanium (IV)-immobilized metal affinity chromatography (Ti-IMAC) material for simultaneous enrichment and separation of neutral and sialyl glycopeptides of a recombinant SARS-CoV-2 S glycoprotein from HEK293 cells. This strategy helps eliminate signal suppression from neutral glycopeptides for the detection of sialyl glycopeptides and improves the glycoform coverage of the S protein. We profiled 19 of its 22 potential N-glycosylated sites with 398 unique glycoforms using the dual-functional Ti-IMAC approach, which exhibited improvement of coverage by 1.6-fold compared to the conventional hydrophilic interaction chromatography (HILIC) glycopeptide enrichment method. We also identified O-linked glycosylation site that was not found using the conventional HILIC approach. In addition, we reported on the identification of mannose-6-phosphate (M6P) glycosylation, which substantially expands the current knowledge of the spike protein’s glycosylation landscape and enables future investigation into the influence of M6P glycosylation of the spike protein on its cell entry.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00216-021-03433-1.

Bifunctional magnetic covalent organic framework for simultaneous enrichment of phosphopeptides and glycopeptides

Protein phosphorylation and glycosylation, which are closely related to various diseases, have been extensively studied recently. Mass spectrometry (MS) based phosphoproteomics and glycoproteomics analysis rely heavily on the pre-treatment. Due to the differences in enrichment conditions, there are still huge challenges in designing and preparing a single affinity material to achieve efficient simultaneous capture and elution of phosphopeptides and glycopeptides. Herein, a novel magnetic covalent organic framework, which was modified with functional molecule 4-(3-(2-(methacryloyloxy)ethyl)-ureido)benzoic acid (MUBA), was designed as a bifunctional enrichment platform for glycopeptides and phosphopeptides. Thanks to the multiple hydrogen bonding interactions between MUBA and hydrogen phosphates, the material possessed excellent enrichment performance for phosphopeptides. In addition, the hydrophilicity of the COF structure and modified molecules endowed this material recognition capability towards glycopeptides based on hydrophilic interaction chromatography. Combining with the inherent properties of COF structure, the established platform achieved simultaneous enrichment of phosphopeptides and glycopeptides with excellent selectivity (1:1:1000 M ratio of α-casein/IgG/BSA), high sensitivity (0.05 fmol/μL α-casein; 0.05 fmol/μL IgG), and good size-exclusion effect (α-casein digests/IgG digests/BSA, 1:1:500). More excitingly, the method was used for the identification of glycopeptides and phosphopeptides from rat liver tissue and the exosomes extracted from liver cancer patients' plasma, proving its specific phosphoproteomics and glycoproteomics study in complex biosamples.

Dual-Functional Ti(IV)-IMAC Material Enables Simultaneous Enrichment and Separation of Diverse Glycopeptides and Phosphopeptides.

Simultaneous enrichment and fractionation of diverse proteins/peptides possessing different post-translational modifications (PTMs) from the same biological samples is highly desirable to reduce sample consumption, avoid complicated sample processing, and enable studies of potential crosstalks between different PTMs. In this work, we report a new approach to enable simultaneous enrichment and separation of glycopeptides, phosphopeptides, and mannose-6-phosphate (M6P) glycopeptides by using a dual-functional Ti(IV)-IMAC material. Moreover, we also made the separation of neutral and sialyl glycopeptides and mono- and multi-phosphopeptides possible by performing different elution processes according to the differences in their electrostatic or hydrophilic properties. These separations are effective and efficient to eliminate the signal suppression from neutral glycopeptides for sialyl glycopeptide detection, allowing separation of mono-phosphopeptides from multi-phosphopeptides, as well as detection of M6P glycopeptides that are free from the abovementioned modifications. This new strategy significantly improves the coverage and identification numbers of glycopeptides, phosphopeptides, and M6P glycopeptides by 1.9, 2.3, and 4.3-fold compared with the conventional method, respectively. This is the first report on simultaneous enrichment and separation of neutral and sialyl glycopeptides, mono- and multi-phosphopeptides, and M6P glycopeptides via dual-functional Ti(IV)- IMAC, revealing novel insights into potential crosstalk among these important PTMs.

Identification of Fucosylated SERPINA1 as a Novel Plasma Marker for Pancreatic Cancer Using Lectin Affinity Capture Coupled with iTRAQ-Based Quantitative Glycoproteomics.

Pancreatic cancer (PC) is an aggressive cancer with a high mortality rate, necessitating the development of effective diagnostic, prognostic and predictive biomarkers for disease management. Aberrantly fucosylated proteins in PC are considered a valuable resource of clinically useful biomarkers. The main objective of the present study was to identify novel plasma glycobiomarkers of PC using the iTRAQ quantitative proteomics approach coupled with Aleuria aurantia lectin (AAL)-based glycopeptide enrichment and isotope-coded glycosylation site-specific tagging, with a view to analyzing the glycoproteome profiles of plasma samples from patients with non-metastatic and metastatic PC and gallstones (GS). As a result, 22 glycopeptides with significantly elevated levels in plasma samples of PC were identified. Fucosylated SERPINA1 (fuco-SERPINA1) was selected for further validation in 121 plasma samples (50 GS and 71 PC) using an AAL-based reverse lectin ELISA technique developed in-house. Our analyses revealed significantly higher plasma levels of fuco-SERPINA1 in PC than GS subjects (310.7 ng/mL v.s. 153.6 ng/mL, p = 0.0114). Elevated fuco-SERPINA1 levels were associated with higher TNM stage (p = 0.024) and poorer prognosis for overall survival (log-rank test, p = 0.0083). The increased plasma fuco-SERPINA1 levels support the utility of this protein as a novel prognosticator for PC.

共价有机骨架功能材料及其在糖肽选择性富集中的应用

蛋白质糖基化是生物体中最重要的翻译后修饰手段之一,糖蛋白/糖肽的有效分离和富集成为目前糖蛋白组学研究的首要问题。对于复杂的生物样本,糖蛋白的数量较少,酶解后大量高丰度非糖基化修饰肽的存在,使得低丰度糖肽的检测更加困难。因此,需要一些手段来有效地富集糖肽以提高其检测丰度,发展高选择性的糖肽富集材料及方法就成为在分子水平上有效地监测糖蛋白或糖肽的重要途径。相对于传统的糖肽富集材料,共价有机骨架材料具有比表面积大和可修饰位点丰富的优点,在糖肽富集领域具有很大的应用潜力。该文制备了一种新型的共价有机骨架材料(O-T-D-COFs),利用1,3,5-三(4-氨苯基)苯和2,5-二甲氧基苯-1,4-二甲醛作为反应单体通过共聚缩合反应生成的席夫碱构成了材料的框架,对合成后的中间体材料进行氧化处理,从而提高材料的富集性能。利用扫描电镜、透射电镜、红外光谱和固体核磁等表征技术对材料的结构进行了表征,并将其应用于糖肽的选择性富集。分别对富集过程的上样条件、淋洗条件、洗脱条件进行了优化,结合质谱检测技术,从人血清免疫球蛋白G酶解液中观察到32个明显的糖肽信号峰。通过模拟复杂样本体系验证材料富集选择性,在人血清免疫球蛋白G和牛血清白蛋白的酶解液混合物摩尔比达到1∶50时,该材料仍然保持了良好的选择性。此外,还考察了材料的检测限、富集容量、回收率等富集性能,及在实际样品中的应用潜力。以人血清免疫球蛋白G为评价对象,O-T-D-COFs具有较低的检测限(2.5 fmol/μL)、较高的富集容量(120 mg/g),及较好的富集回收率(103.5%±6.6%、101.5%±10.4%)。在血清样品中富集到来自53个N-糖蛋白中的86个N-糖肽序列,并鉴定到了94个N-糖基化位点。这些结果都表明,该材料在糖肽富集领域有较好的应用前景。

Maternal and Child Supplementation With Small-Quantity Lipid-Based Nutrient Supplements Increases Child HDL Cholesterol Efflux Capacity

ObjectivesThe purpose of this secondary outcome analysis is to investigate whether small-quantity lipid-based nutrient supplements (SQ-LNS) alters lipid, protein or glycan composition, or the cholesterol efflux capacity (CEC), of high-density lipoprotein (HDL) particles in children in the International Lipid-Based Nutrient Supplements (iLiNS) DYAD trial in Ghana. MethodsPlasma samples were obtained from a subcohort of 80 children at 18 months of age from the iLiNS-DYAD-Ghana trial. Mothers were randomized to either iron and folic acid (IFA) in pregnancy and 200 mg/d calcium for 6 months postpartum or SQ-LNS (pregnancy and 6 months postpartum). Children in the SQ-LNS group received SQ-LNS from 6 to 18 months while children in the IFA group did not receive supplements. HDL was isolated from plasma by sequential ultracentrifugation followed by size-exclusion chromatography. Assay of cholesterol efflux was performed in vitro, and glycoproteomic and lipidomic composition were analyzed by mass spectrometry. The primary analysis was a comparison of the effects of intervention groups on HDL lipidome, proteome, and CEC. In the exploratory analysis, we compared the enrichment of glycopeptides in measured HDL-associated proteins between groups. ResultsMean (±SD) HDL CEC was higher among children in the SQ-LNS vs. IFA group (20.9 ± 4.1% vs. 19.4 ± 3.3%; one-tailed p = 0.038). We found no differences in HDL lipidomic or proteomic composition between groups. ConclusionsPrenatal and postnatal SQ-LNS may improve the CEC of child HDL particles. These improvements may have a potential impact on child health outcomes. Funding SourcesSupported by Bill & Melinda Gates Foundation grant to the University of California, Davis.

Glycoproteomic analysis of the changes in protein N-glycosylation during neuronal differentiation in human-induced pluripotent stem cells and derived neuronal cells

N-glycosylation of glycoproteins, a major post-translational modification, plays a crucial role in various biological phenomena. In central nervous systems, N-glycosylation is thought to be associated with differentiation and regeneration; however, the state and role of N-glycosylation in neuronal differentiation remain unclear. Here, we conducted sequential LC/MS/MS analyses of tryptic digest, enriched glycopeptides, and deglycosylated peptides of proteins derived from human-induced pluripotent stem cells (iPSCs) and iPSC-derived neuronal cells, which were used as a model of neuronal differentiation. We demonstrate that the production profiles of many glycoproteins and their glycoforms were altered during neuronal differentiation. Particularly, the levels of glycoproteins modified with an N-glycan, consisting of five N-acetylhexosamines, three hexoses, and a fucose (HN5H3F), increased in dopaminergic neuron-rich cells (DAs). The N-glycan was deduced to be a fucosylated and bisected biantennary glycan based on product ion spectra. Interestingly, the HN5H3F-modified proteins were predicted to be functionally involved in neural cell adhesion, axon guidance, and the semaphorin-plexin signaling pathway, and protein modifications were site-selective and DA-selective regardless of protein production levels. Our integrated method for glycoproteome analysis and resultant profiles of glycoproteins and their glycoforms provide valuable information for further understanding the role of N-glycosylation in neuronal differentiation and neural regeneration.

Highly Efficient Enrichment of O-GalNAc Glycopeptides by Using Immobilized Metal Ion Affinity Chromatography.

Proteomics analysis of O-GalNAc glycosylation is important for the screening of biomarkers and the assessment of therapeutic responses. However, its analysis still faces challenges due to the poor performance of currently available enrichment methods. In this study, an enrichment method was established on the basis of Ti-IMAC(IV) materials, which could enrich the intact O-GalNAc glycopeptides via both the hydrophilic interaction and affinity interaction. This method enabled nearly 200 intact O-GalNAc glycopeptides identified from only 0.1 μL of human serum. This was nearly 2-fold different from that of the HILIC method. An in-depth analysis of the O-GalNAc glycosylation was performed, and 2093 intact glycopeptides were identified from 7.2 μL of human serum samples. This is the largest O-GalNAc glycosylation database of human serum from a trace amount of sample. Furthermore, 52 significantly changed intact O-GalNAc glycopeptides were determined by the quantitative analysis of hepatocellular carcinoma (HCC) and control serum samples, indicating the potential applications of this enrichment method in biomarker discovery.

Mass Spectrometry-Based Glycoproteomics and Prostate Cancer.

Aberrant glycosylation has long been known to be associated with cancer, since it is involved in key mechanisms such as tumour onset, development and progression. This review will focus on protein glycosylation studies in cells, tissue, urine and serum in the context of prostate cancer. A dedicated section will cover the glycoforms of prostate specific antigen, the molecule that, despite some important limitations, is routinely tested for helping prostate cancer diagnosis. Our aim is to provide readers with an overview of mass spectrometry-based glycoproteomics of prostate cancer. From this perspective, the first part of this review will illustrate the main strategies for glycopeptide enrichment and mass spectrometric analysis. The molecular information obtained by glycoproteomic analysis performed by mass spectrometry has led to new insights into the mechanism linking aberrant glycosylation to cancer cell proliferation, migration and immunoescape.

Automated Intact Glycopeptide Enrichment Method Facilitating Highly Reproducible Analysis of Serum Site-Specific N-Glycoproteome.

Bottom-up proteomics has been increasingly applied in clinical research to study the disease pathophysiology and to discover disease biomarkers. However, glycoproteomic analysis always requires tedious experimental steps for intact glycopeptide enrichment, which has been the technique bottleneck for large-scale analysis of clinical samples. Herein, we developed an automated glycopeptide enrichment method for the analysis of serum site-specific N-glycoproteome. This automated method allowed for processing one sample within 20 min. It showed higher enrichment specificity, more intact glycopeptide identifications, and better quantitative reproducibility than the traditional manual method using microtip enrichment devices. We further applied this method to investigate the serum site-specific N-glycosylation changes between four patients with pancreatic cancer and seven healthy controls. The principal component analysis of intact N-glycopeptides showed good clustering across cancer and normal groups. Furthermore, we found that the site-specific glycoforms, monofucosylated and nonsialylated oligosaccharides, on IgG1 site 180 expressed a significant decrease in pancreatic cancer patients compared to healthy controls. Together, the automated method is a powerful tool for site-specific N-glycoproteomic analysis of complex biological samples, and it has great potential for clinical utilities.