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Protein glycosylation is implicated in a wide array of diseases, yet glycoprotein analysis remains elusive owing to the extreme heterogeneity of glycans, including microheterogeneity of some of the glycosites (amino acid residues). Various mass spectrometry (MS) strategies have proven tremendously successful for localizing and identifying glycans, typically utilizing a bottom-up workflow in which glycoproteins are digested to create glycopeptides to facilitate analysis. An emerging alternative is top-down MS that aims to characterize intact glycoproteins to allow precise identification and localization of glycans. The most comprehensive characterization of intact glycoproteins requires integration of a suitable separation method and high performance tandem mass spectrometry to provide both protein sequence information and glycosite localization. Here, we couple ultraviolet photodissociation and hydrophilic interaction chromatography with high resolution mass spectrometry to advance the characterization of intact glycoproteins ranging from 15 to 34 kDa, offering site localization of glycans, providing sequence coverages up to 93%, and affording relative quantitation of individual glycoforms.

Mild cognitive impairment (MCI) is an early stage of memory loss that affects cognitive abilities with the aging of individuals, such as language or visual/spatial comprehension. MCI is considered a prodromal phase of more complicated neurodegenerative diseases such as Alzheimer's. Therefore, accurate diagnosis and better understanding of the disease prognosis will facilitate prevention of neurodegeneration. However, the existing diagnostic methods fail to provide precise and well-timed diagnoses, and the pathophysiology of MCI is not fully understood. Alterations of the serum N-glycoproteome expression could represent an essential contributor to the overall pathophysiology of neurodegenerative diseases and be used as a potential marker to assess MCI diagnosis using less invasive procedures. In this approach, we identified N-glycopeptides with different expressions between healthy and MCI patients from serum glycoproteins. Seven of the N-glycopeptides showed outstanding AUC values, among them the antithrombin-III Asn224 + 4-5-0-2 with an AUC value of 1.00 and a p value of 0.0004. According to proteomics and ingenuity pathway analysis (IPA), our data is in line with recent publications, and the glycoproteins carrying the identified N-sites play an important role in neurodegeneration., (© 2024 Wiley‐VCH GmbH.)

Rationale: A general N-glycoproteomics analysis pipeline has been established for characterization of mutation-related gain-of-glycosylation (GoG) at intact N-glycopeptide molecular level, generating comprehensive site and structure information of N-glycosylation., Methods: This study focused on mutation-originated GoG using a mass spectrometry-based N-glycoproteomics analysis workflow. In brief, GoG intact N-glycopeptide databases were built, consisting of 2701 proteins (potential GoG N-glycosites and amino acids derived from MUTAGEN, VARIANT and VAR_SEQ in UniProt) and 6709 human N-glycans (≤50 sequence isomers per monosaccharide composition). We employed the site- and structure-specific N-glycoproteomics workflow utilizing intact N-glycopeptides search engine GPSeeker to identify GoG intact N-glycopeptides from parental breast cancer stem cells (MCF-7 CSCs) and adriamycin-resistant breast cancer stem cells (MCF-7/ADR CSCs)., Results: With the criteria of spectrum-level false discovery rate control of ≤1%, we identified 87 and 94 GoG intact N-glycopeptides corresponding to 37 and 35 intact N-glycoproteins from MCF-7 CSCs and MCF-7/ADR CSCs, respectively. Micro-heterogeneity and macro-heterogeneity of N-glycosylation from GoG intact N-glycoproteins with VAR_SEQ and VARIANT were found in both MCF-7 CSCs and MCF-7/ADR CSCs systems., Conclusions: The integration of site- and structure-specific N-glycoproteomics approach, conjugating with GoG characterization, provides a universal workflow for revealing comprehensive N-glycosite and N-glycan structure information of GoG. The analysis of mutation-originated GoG can be extended to GoG characterization of other N-glycoproteome systems including complex clinical tissues and body fluids., (© 2024 John Wiley & Sons Ltd.)

Protein glycosylation is the co- and/or post-translational modification of proteins with oligosaccharides (glycans). This process is not template based and can introduce a heterogeneous set of glycan modifications onto substrate proteins. Glycan structures preserve biomolecular information from the cell, with glycoproteins from different cell types and tissues displaying distinct patterns of glycosylation. Several decades of research have revealed that glycan structures also differ between normal physiology and disease. This suggests that the information stored in glycoproteins and glycans can be utilized for disease diagnosis and monitoring. Methods that enable sensitive and site-specific measurement of protein glycosylation in clinical settings, such as nano-flow liquid chromatography tandem mass spectrometry, are therefore essential. The purpose of this perspective is to discuss recent advances in mass spectrometry and the potential of these advances to facilitate the detection and monitoring of disease-specific glycoprotein glycoforms. Glycoproteomics, the system-wide characterization of glycoprotein identity inclusive of site-specific characterization of carbohydrate modifications on proteins, and glycomics, the characterization of glycan structures, will be discussed in this context. Quantitative measurement of glycopeptide markers via parallel reaction monitoring is highlighted. The development of promising glycopeptide markers for autoimmune disease, liver disease, and liver cancer is discussed. Synthetic glycopeptide standards, ambient ionization mass spectrometry, and consideration of glyco-biomarkers in two- and three-dimensional space within tissue will be critical to the advancement of this field. The authors envision a future in which glycoprotein mass spectrometry workflows will be integrated into clinical settings, to aid in the rapid diagnosis and monitoring of disease., (© 2024 John Wiley & Sons Ltd.)

Salt stress is one of the significant environmental stresses that severely affect plant growth and development. Here, we report quantitative N-glycoproteomics characterization of differential N-glycosylation in Sorghum bicolor under low, median and high salinity stress. 21,621 intact N-glycopeptides coming from the combination of 127 N-glycan structures on 6574 N-glycosites from 5321 proteins were identified; differential N-glycosylation was observed for 682 N-glycoproteins which are mainly involved in the pathways of biosynthesis of secondary metabolites, biosynthesis of amino acids and several metabolic pathways. 41 N-glycan structures modifying on 338 N-glycopeptides from 122 glycoproteins were co-quantified and deregulated under at least one salt stress, including enzymes of energy production and carbohydrate metabolisms, cell wall organization related proteins, glycosyltransferases and so on. Intriguingly, with increasing salt concentration, there was an increase in the percentage of complex N-glycans on the altered N-glycopeptides. Furthermore, the observation of glycoproteins with distinct salt sensitivity is noteworthy, particularly the upregulated hyposensitive glycoproteins that predominantly undergo complex N-glycan modification. This is the first N-glycoproteome description of salt stress response at the intact N-glycopeptide level in sorghum and a further validation of data reported here would likely provide deeper insights into the stress physiology of this important crop plant., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

N- linked glycosylation is a common posttranslational modification of proteins that results in macroheterogeneity of the modification site. However, unlike simpler modifications, N- glycosylation introduces an additional layer of complexity with tens of thousands of possible structures arising from various dimensions, including different monosaccharide compositions, sequence structures, linking structures, isomerism, and three-dimensional conformations. This results in additional microheterogeneity of the modification site of N- glycosylation, i.e., the same N- glycosylation site can be modified with different glycans with a certain stoichiometric ratio. N- glycosylation regulates the structure and function of N- glycoproteins in a site- and structure-specific manner, and differential expression of N- glycosylation under disease conditions needs to be characterized through site- and structure-specific quantitative analysis. Numerous advanced methods ranging from sample preparation to mass spectrum analysis have been developed to distinguish N-glycan structures. Chemical derivatization of monosaccharides, online liquid chromatography separation and ion mobility spectrometry enable the physical differentiation of samples. Tandem mass spectrometry further analyzes the macro/microheterogeneity of intact N- glycopeptides through the analysis of fragment ions. Moreover, the development of search engines and AI-based software has enhanced our understanding of the dissociation patterns of intact N- glycopeptides and the clinical significance of differentially expressed intact N- glycopeptides. With the help of these modern methods, structure-specific N- glycoproteomics has become an important tool with extensive applications in the biomedical field.

Protein glycosylation is a ubiquitous process observed across all domains of life. Within the human pathogen Acinetobacter baumannii , O -linked glycosylation is required for virulence; however, the targets and conservation of glycosylation events remain poorly defined. In this work, we expand our understanding of the breadth and site specificity of glycosylation within A. baumannii by demonstrating the value of strain specific glycan electron-transfer/higher-energy collision dissociation (EThcD) triggering for bacterial glycoproteomics. By coupling tailored EThcD-triggering regimes to complementary glycopeptide enrichment approaches, we assessed the observable glycoproteome of three A. baumannii strains (ATCC19606, BAL062, and D1279779). Combining glycopeptide enrichment techniques including ion mobility (FAIMS), metal oxide affinity chromatography (titanium dioxide), and hydrophilic interaction liquid chromatography (ZIC-HILIC), as well as the use of multiple proteases (trypsin, GluC, pepsin, and thermolysis), we expand the known A. baumannii glycoproteome to 33 unique glycoproteins containing 42 glycosylation sites. We demonstrate that serine is the sole residue subjected to glycosylation with the substitution of serine for threonine abolishing glycosylation in model glycoproteins. An A. baumannii pan-genome built from 576 reference genomes identified that serine glycosylation sites are highly conserved. Combined this work expands our knowledge of the conservation and site specificity of A. baumannii O -linked glycosylation.

Mucin-domain glycoproteins are characterized by their high density of glycosylated serine and threonine residues, which complicates their analysis by mass spectrometry. The dense glycosylation renders the protein backbone inaccessible to workhorse proteases like trypsin, the vast heterogeneity of glycosylation often results in ion suppression from unmodified peptides, and search algorithms struggle to confidently analyze and site-localize O-glycosites. We have made a number of advances to address these challenges, rendering mucinomics possible for the first time. Here, we summarize these contributions and provide a detailed protocol for mass spectrometric analysis of mucin-domain glycoproteins. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Enrichment of mucin-domain glycoproteins Basic Protocol 2: Enzymatic digestion of mucin-domain glycoprotein(s) Basic Protocol 3: Mass spectrometry data collection for O-glycopeptides Basic Protocol 4: Mass spectrometry data analysis of O-glycopeptides., (© 2024 Wiley Periodicals LLC.)

N-glycosylation is one of the most universal and complex protein post-translational modifications (PTMs), and it is involved in many physiological and pathological activities. Owing to the low abundance of N-glycoproteins, enrichment of N-glycopeptides for mass spectrometry analysis usually requires a large amount of peptides. Additionally, oocyte protein N-glycosylation has not been systemically characterized due to the limited sample amount. Here, we developed a glycosylation enrichment method based on lectin and a single-pot, solid-phase-enhanced sample preparation (SP3) technology, termed lectin-based SP3 technology (LectinSP3). LectinSP3 immobilized lectin on the SP3 beads for N-glycopeptide enrichment. It could identify over 1100 N-glycosylation sites and 600 N-glycoproteins from 10 μg of mouse testis peptides. Furthermore, using the LectinSP3 method, we characterized the N-glycoproteome of 1000 mouse oocytes in three replicates and identified a total of 363 N-glycosylation sites from 215 N-glycoproteins. Bioinformatics analysis revealed that these oocyte N-glycoproteins were mainly enriched in cell adhesion, fertilization, and sperm-egg recognition. Overall, the LectinSP3 method has all procedures performed in one tube, using magnetic beads. It is suitable for analysis of a low amount of samples and is expected to be easily adaptable for automation. In addition, our mouse oocyte protein N-glycosylation profiling could help further characterize the regulation of oocyte functions.

Extracellular vesicles (EVs) are lipid bilayer-enclosed particles that can be released by all type of cells. Whereas, as one of the most common post-translational modifications, glycosylation plays a vital role in various biological functions of EVs, such as EV biogenesis, sorting, and cellular recognition. Nevertheless, compared with studies on RNAs or proteins, those investigating the glycoconjugates of EVs are limited. An in-depth investigation of N-glycosylation of EVs can improve the understanding of the biological functions of EVs and help to exploit EVs from different perspectives. The general focus of studies on glycosylation of EVs primarily includes isolation and characterization of EVs, preparation of glycoproteome/glycome samples and MS analysis. However, the low content of EVs and non-standard separation methods for downstream analysis are the main limitations of these studies. In this review, we highlight the importance of glycopeptide/glycan enrichment and derivatization owing to the low abundance of glycoproteins and the low ionization efficiency of glycans. Diverse fragmentation patterns and professional analytical software are indispensable for analysing glycosylation via MS. Altogether, this review summarises recent studies on glycosylation of EVs, revealing the role of EVs in disease progression and their remarkable potential as biomarkers., (© 2023 Wiley‐VCH GmbH.)

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., (© 2024 John Wiley & Sons Ltd.)

A comprehensive analysis of site-specific protein O-glycosylation is hindered by the absence of a consensus O-glycosylation motif, the diversity of O-glycan structures, and the lack of a universal enzyme that cleaves attached O-glycans. Here, we report the development of a robust O-glycoproteomic workflow for analyzing complex biological samples by combining four different strategies: removal of N-glycans, complementary digestion using O-glycoprotease (IMPa) with/without another protease, glycopeptide enrichment, and mass spectrometry with fragmentation of glycopeptides using stepped collision energy. Using this workflow, we cataloged 474 O-glycopeptides on 189 O-glycosites derived from 79 O-glycoproteins from human plasma. These data revealed O-glycosylation of several abundant proteins that have not been previously reported. Because many of the proteins that contained unannotated O-glycosylation sites have been extensively studied, we wished to confirm glycosylation at these sites in a targeted fashion. Thus, we analyzed selected purified proteins (kininogen-1, fetuin-A, fibrinogen, apolipoprotein E, and plasminogen) in independent experiments and validated the previously unknown O-glycosites., Competing Interests: Declaration of interests The authors declare no competing interests, (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Hydrophilic interaction liquid chromatography (HILIC) is widely used for glycopeptide enrichment in shot-gun glycoproteomics to enhance the glycopeptide signal and minimize the ionization competition of peptides. In this work, we have developed a novel hydrophilic material (glycoHILIC) based on glycopeptides and peptides to provide hydrophilic properties. GlycoHILIC was synthesized by oxidizing cotton and then reacting the resulting aldehyde with the N-terminus of the glycopeptide or peptide by reductive amination. Due to the large amount of hydrophilic carbohydrates and hydrophilic amino acids contained in glycopeptides, glycoHILIC showed significantly better enrichment of glycopeptides than cotton itself. Our results demonstrate that glycoHILIC has high selectivity, a low detection limit, and good stability. Over 257 unique N-linked glycosylation sites in 1477 intact N-glycopeptides from 146 glycoproteins were identified from 1 μL of human serum using glycoHILIC. Serum analysis of pancreatic cancer patients found that 38 N-glycopeptides among 21 glycoproteins changed significantly, of which 7 N-glycopeptides increased and 31 N-glycopeptides decreased. These results demonstrate that glycoHILIC can be used for glycopeptide enrichment and analysis.

Alpha-1,6 fucosylation of N-glycans (core fucosylation, CF) represents a unique form of N-glycans and is widely involved in disease progression. In order to accurately identify CF glycoproteins, several approaches have been developed based on sequential cleavage with different glycosidases to truncate the N-glycans. Since multi-step sample treatments may introduce quantitation bias and affect the practicality of these approaches in large-scale applications. Here, we systematically evaluated the performance of the single-step treatment of intact glycopeptides by endoglycosidase F3 for CF glycoproteome. The single-step truncation (SST) strategy demonstrated higher quantitative stability and higher efficiency compared with previous approaches. The strategy was further practiced on both cell lines and serum samples. The dysregulation of CF glycopeptides between preoperative and postoperative serum from patients with pancreatic ductal adenocarcinoma was revealed, and the CF modifications of BCHE_N369, CDH5_N112 and SERPIND1_N49 were found to be potential prognostic markers. This study thus provides an efficient solution for large-scale quantitative analysis of the CF glycoproteome., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Mass spectrometry (MS) can unlock crucial insights into the intricate world of glycosylation analysis. Despite its immense potential, the qualitative and quantitative analysis of isobaric glycopeptide structures remains one of the most daunting hurdles in the field of glycoproteomics. The ability to distinguish between these complex glycan structures poses a significant challenge, hindering our ability to accurately measure and understand the role of glycoproteins in biological systems. A few recent publications described the use of collision energy (CE) modulation to improve structural elucidation, especially for qualitative purposes. Different linkages of glycan units usually demonstrate different stabilities under CID/HCD fragmentation conditions. Fragmentation of the glycan moiety produces low molecular weight ions (oxonium ions) that can serve as a structure-specific signature for specific glycan moieties; however, the specificity of these fragments has never been examined closely. Here, we particularly focused on N-glycoproteomics analysis and investigated fragmentation specificity using synthetic stable isotope-labeled N-glycopeptide standards. These standards were isotopically labeled at the reducing terminal GlcNAc, which allowed us to resolve fragments produced by the oligomannose core moiety and fragments generated from outer antennary structures. Our research identified the potential for false-positive structure assignments due to the occurrence of "Ghost" fragments resulting from single glyco unit rearrangement or mannose core fragmentation within the collision cell. To mitigate this issue, we have established a minimal intensity threshold for these fragments to prevent misidentification of structure-specific fragments in glycoproteomics analysis. Our findings provide a crucial step forward in the quest for more accurate and reliable glycoproteomics measurements.

Both N-linked glycosylation and O-linked glycosylation play essential roles in the onset and progression of various diseases including cancer, and N-/O-linked site-specific glycans have been proven to be promising biomarkers for the discrimination of cancer. However, the micro-heterogeneity and low abundance nature of N-/O-linked glycosylation, as well as the time-consuming and tedious procedures for the enrichment of O -linked intact glycopeptides, pose great challenges for their efficient and accurate characterization. In this study, we developed an integrated platform for the simultaneous enrichment and characterization of N- and O-linked intact glycopeptides from the same serum sample. By fine-tuning the experimental conditions, we demonstrated that this platform allowed the selective separation of N- and O-linked intact glycopeptides into two fractions, with 85.1% O-linked intact glycopeptides presented in the first fraction and 93.4% N-linked intact glycopeptides presented in the second fraction. Determined with high reproducibility, this platform was further applied to the differential analysis of serum samples of gastric cancer and health control, which revealed 17 and 181 significantly changed O-linked and N-linked intact glycopeptides. Interestingly, five glycoproteins containing both significant regulation of N- and O-glycosylation were observed, hinting potential co-regulation of different types of glycosylation during tumor progress. In summary, this integrated platform opened a potentially useful avenue for the global analysis of protein glycosylation and can serve as a useful tool for the characterization of N-/O-linked intact glycopeptides at the proteomics scale.

Rheumatoid arthritis (RA) is a typical autoimmune disease characterized by synovial inflammation, synovial tissue hyperplasia, and destruction of bone and cartilage. Protein glycosylation plays key roles in the pathogenesis of RA but in-depth glycoproteomics analysis of synovial tissues is still lacking. Here, by using a strategy to quantify intact N-glycopeptides, we identified 1260 intact N-glycopeptides from 481 N-glycosites on 334 glycoproteins in RA synovium. Bioinformatics analysis revealed that the hyper-glycosylated proteins in RA were closely linked to immune responses. By using DNASTAR software, we identified 20 N-glycopeptides whose prototype peptides were highly immunogenic. We next calculated the enrichment scores of nine types of immune cells using specific gene sets from public single-cell transcriptomics data of RA and revealed that the N-glycosylation levels at some sites, such as IGSF10_N2147, MOXD2P_N404, and PTCH2_N812, were significantly correlated with the enrichment scores of certain immune cell types. Furthermore, we showed that aberrant N-glycosylation in the RA synovium was related to increased expression of glycosylation enzymes. Collectively, this work presents, for the first time, the N-glycoproteome of RA synovium and describes immune-associated glycosylation, providing novel insights into RA pathogenesis., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Recent advances in analytical techniques provide the opportunity to quantify even low-abundance glycopeptides derived from complex biological mixtures, allowing for the identification of glycosylation differences between healthy samples and those derived from disease states. Herein, we discuss the sample preparation procedures and the mass spectrometry (MS) strategies that have facilitated glycopeptide quantification, as well as the standards used for glycopeptide quantification. For sample preparation, various glycopeptide enrichment methods are summarized including the columns used for glycopeptide separation in liquid chromatography separation. For MS analysis strategies, MS1 level-based quantification and MS2 level-based quantification are described, either with or without labeling, where we have covered isotope labeling, TMT/iTRAQ labeling, data dependent acquisition, data independent acquisition, multiple reaction monitoring, and parallel reaction monitoring. The strengths and weaknesses of these methods are compared, particularly those associated with the figures of merit that are important for clinical biomarker studies and the pathological and functional studies of glycoproteins in various diseases. Possible future developments for glycopeptide quantification are discussed., (© 2022 John Wiley & Sons Ltd.)

Tweetable abstract Bottom-up glycoproteomics combined with top-down strategy allows direct analysis of glycoform-mapped glycosylation and its glycans by high-resolution mass spectrometry.

Advances in mass spectrometry instrumentation, methods development, and bioinformatics have greatly improved the ease and accuracy of site-specific, quantitative glycoproteomics analysis. Data-dependent acquisition is the most popular method for identification and quantification of glycopeptides; however, complete coverage of glycosylation site glycoforms remains elusive with this method. Targeted acquisition methods improve the precision and accuracy of quantification, but at the cost of throughput and discoverability. Data-independent acquisition (DIA) holds great promise for more complete and highly quantitative site-specific glycoproteomics analysis, while maintaining the ability to discover novel glycopeptides without prior knowledge. We review additional features that can be used to increase selectivity and coverage to the DIA workflow: retention time modeling, which would simplify the interpretation of complex tandem mass spectra, and ion mobility separation, which would maximize the sampling of all precursors at a giving chromatographic retention time. The instrumentation and bioinformatics to incorporate these features into glycoproteomics analysis exist. These improvements in quantitative, site-specific analysis will enable researchers to assess glycosylation similarity in related biological systems, answering new questions about the interplay between glycosylation state and biological function., (© 2021 John Wiley & Sons Ltd.)

As a key regulator of many biological processes, glycosylation is an essential post-translational modification (PTM) in the living system. Over 50% of human proteins are known to be glycosylated. Alterations in glycoproteins are directly linked to many diseases, making it crucial to understand system-wide glycosylation changes. The majority of known glycoproteins are from plasma membrane; however, glycosylation is a dynamic process that occurs throughout multiple subcellular organelles and involves sets of enzymes, chaperones, transporters, and sugar donor molecules. Many glycoproteins are expressed not only in plasma membranes but also in subcellular organelles. Here, we developed a mass-spectrometry-based quantitative workflow for the system-wide N-glycoproteomic analysis of membrane and cytosolic proteins extracted using a MEM-PER kit. The kit facilitates the extraction and solubilization of both membrane and cytosolic proteins in a simple, efficient, and reproducible manner. We analyzed the K562 cell line and mouse liver tissue to evaluate this approach. A total of 934 glycosites, 5154 glycopeptides, and 536 glycoproteins from the K562 cell line and a total of 1449 glycosites, 7549 glycopeptides, and 660 glycoproteins from mouse liver tissue were identified. This simple and reproducible approach provides a unique way to understand system-wide glycosylation in biological processes and enables the identification and quantitation of glycan profiles at glycosylation sites in proteins.

Protein glycosylation that mediates interactions among viral proteins, host receptors, and immune molecules is an important consideration for predicting viral antigenicity. Viral spike proteins, the proteins responsible for host cell invasion, are especially important to be examined. However, there is a lack of consensus within the field of glycoproteomics regarding identification strategy and false discovery rate (FDR) calculation that impedes our examinations. As a case study in the overlap between software, here as a case study, we examine recently published SARS-CoV-2 glycoprotein datasets with four glycoproteomics identification software with their recommended protocols: GlycReSoft, Byonic, pGlyco2, and MSFragger-Glyco. These software use different Target-Decoy Analysis (TDA) forms to estimate FDR and have different database-oriented search methods with varying degrees of quantification capabilities. Instead of an ideal overlap between software, we observed different sets of identifications with the intersection. When clustering by glycopeptide identifications, we see higher degrees of relatedness within software than within glycosites. Taking the consensus between results yields a conservative and non-informative conclusion as we lose identifications in the desire for caution; these non-consensus identifications are often lower abundance and, therefore, more susceptible to nuanced changes. We conclude that present glycoproteomics softwares are not directly comparable, and that methods are needed to assess their overall results and FDR estimation performance. Once such tools are developed, it will be possible to improve FDR methods and quantify complex glycoproteomes with acceptable confidence, rather than potentially misleading broad strokes.

Precision mapping of glycans at structural and site-specific level is still one of the most challenging tasks in the glycobiology field. Here, we describe a modularization strategy for de novo interpretation of N-glycan structures on intact glycopeptides using tandem mass spectrometry. An algorithm named StrucGP is also developed to automate the interpretation process for large-scale analysis. By dividing an N-glycan into three modules and identifying each module using distinct patterns of Y ions or a combination of distinguishable B/Y ions, the method enables determination of detailed glycan structures on thousands of glycosites in mouse brain, which comprise four types of core structure and 17 branch structures with three glycan subtypes. Owing to the database-independent glycan mapping strategy, StrucGP also facilitates the identification of rare/new glycan structures. The approach will be greatly beneficial for in-depth structural and functional study of glycoproteins in the biomedical research., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Rationale: While high-throughput proteomic methods have been widely applied to monoclonal antibodies and human immunoglobulin gamma (IgG) samples, less information is available on porcine IgG. As pigs are considered one of the most suitable species for xenotransplantation, it is important to characterize IgG amino acid sequences and glycosylation profiles, which is the focus of this study., Methods: Three different purified porcine IgG samples, including wild-type and knockout species, were digested with trypsin and enriched for glycopeptides. Digestion mixtures were spiked with a mixture of six standard peptides. Analysis was performed using electrospray ionization liquid chromatography-tandem mass spectrometry (MS/MS) in standard MS/MS data-dependent acquisition mode on a hybrid triple quadrupole time-of-flight mass spectrometer., Results: To facilitate the classification of subtypes detected experimentally, UniprotKB database entries were organized using comparative alignment scores. Sequences were grouped based on 11 different subtypes as translated from GenBank entries. Proteomic searches were accomplished automatically using specialized software, whereas glycoprotein searches were performed manually by monitoring the extracted chromatograms of diagnostic MS/MS glycan fragments and studying their corresponding mass spectra; 40-50 non-glycosylated peptides and 4-5 glycosylated peptides were detected in each sample, with several glycoforms per sequence., Conclusions: Proteomic analysis of porcine IgG is complicated by factors such as the presence of several subtypes, redundant heavy chain (HC) sequences in protein databases, and the lack of consistent cross-referencing between databases. Aligning and comparing HC sequences were necessary to eliminate redundancy. This study highlights the complexity of pig IgG and shows the importance of MS in proteomics and glycoproteomics., (© 2021 John Wiley & Sons Ltd.)

Sparkling wine is an alcoholic beverage enjoyed around the world. The sensory properties of sparkling wine depend on a complex interplay between the chemical and biochemical components in the final product. Glycoproteins have been linked to positive and negative qualities in sparkling wine, but the glycosylation profiles of sparkling wine have not been previously investigated in detail. We analyzed the glycoproteome of sparkling wines using protein- and glycopeptide-centric approaches. We developed an automated workflow that created ion libraries to analyze sequential window acquisition of all theoretical mass spectra data-independent acquisition mass spectrometry data based on glycopeptides identified by Byonic (Protein Metrics; version 2.13.17). We applied our workflow to three pairs of experimental sparkling wines to assess the effects of aging on lees and of different yeast strains used in the liqueur de tirage for secondary fermentation. We found that aging a cuvée on lees for 24 months compared with 8 months led to a dramatic decrease in overall protein abundance and an enrichment in large glycans at specific sites in some proteins. Secondary fermentation of a Riesling wine with Saccharomyces cerevisiae yeast strain Siha4 produced more yeast proteins and glycoproteins than with S. cerevisiae yeast strain DV10. The abundance and glycosylation profiles of grape glycoproteins were also different between grape varieties. To our knowledge, this work represents the first in-depth study into protein- and peptide-specific glycosylation in sparkling wines and describes a quantitative glycoproteomic sequential window acquisition of all theoretical mass spectra/data-independent acquisition workflow that is broadly applicable to other sample types., Competing Interests: Conflict of interest Christopher H. Caboche was supported by a Wine Australia Research Scholarship Ph1903. The other authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Glycosylation is a prevalent, yet heterogeneous modification with a broad range of implications in molecular biology. This heterogeneity precludes enrichment strategies that can be universally beneficial for all glycan classes. Thus, choice of enrichment strategy has profound implications on experimental outcomes. Here we review common enrichment strategies used in modern mass spectrometry-based glycoproteomic experiments, including lectins and other affinity chromatographies, hydrophilic interaction chromatography and its derivatives, porous graphitic carbon, reversible and irreversible chemical coupling strategies, and chemical biology tools that often leverage bioorthogonal handles. Interest in glycoproteomics continues to surge as mass spectrometry instrumentation and software improve, so this review aims to help equip researchers with the necessary information to choose appropriate enrichment strategies that best complement these efforts., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Complex protein glycosylation occurs through biosynthetic steps in the secretory pathway that create macro- and microheterogeneity of structure and function. Required for all life forms, glycosylation diversifies and adapts protein interactions with binding partners that underpin interactions at cell surfaces and pericellular and extracellular environments. Because these biological effects arise from heterogeneity of structure and function, it is necessary to measure their changes as part of the quest to understand nature. Quite often, however, the assumption behind proteomics that posttranslational modifications are discrete additions that can be modeled using the genome as a template does not apply to protein glycosylation. Rather, it is necessary to quantify the glycosylation distribution at each glycosite and to aggregate this information into a population of mature glycoproteins that exist in a given biological system. To date, mass spectrometric methods for assigning singly glycosylated peptides are well-established. But it is necessary to quantify glycosylation heterogeneity accurately in order to gauge the alterations that occur during biological processes. The task is to quantify the glycosylated peptide forms as accurately as possible and then apply appropriate bioinformatics algorithms to the calculation of micro- and macro-similarities. In this review, we summarize current approaches for protein quantification as they apply to this glycoprotein similarity problem., Competing Interests: Conflicts of interest The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

O -Glycoprotein analysis has been historically challenging due, in part, to a dearth of available enzymes active in the release of O -glycans. Moreover, chemical releasing methods, such as β-elimination/Michael addition, are not specific to O -glycan release and can also eliminate phosphoryl substitutions. Both of these events leave behind deaminated serine and threonine and thus can lead to ambiguous structural conclusions. Recently, the O -protease OpeRATOR, derived from intestinal bacteria and expressed in Escherichia coli , has become commercially available. The digestion of O -glycoprotein yields O -glycopeptides cleaved at the N- terminal end of serine and threonine, with O -glycan remaining intact. The enzyme has a broad substrate specificity and includes mammalian cores 1-8. However, OpeRATOR is not fully active toward sialylated glycoproteins, and it has been suggested that this acidic residue be removed prior to digestion, thus sacrificing structural information. In this study, we investigated the performance of OpeRATOR under a range of conditions, including buffer selection, varying pH, sialic acid modification, and digestion temperature, in order to optimize the enzymatic activity, with a special emphasis on sialylated glycosites. Conditions derived in this work facilitate the OpeRATOR digestion of fully sialylated O -glycopeptides that are mass tagged to identify the sialyl linkage, thus facilitating the analysis of these charged O -glycopeptides, which are often important in biological processes.

Tn-antigen (Tn), a single N -acetylgalactosamine (GalNAc) monosaccharide attached to protein Ser/Thr residues, is found on most cancer yet rarely detected in adult normal tissues as reported in previous studies, featuring it as one of the most distinctive signatures of cancer. Although it is important in cancer, Tn modified glycoproteins are not entirely clear owing to the lack of a suitable method. Knowing the Tn-glycosylated proteins and glycosylation sites are essential to the prevention, diagnosis, and therapy of cancer associated with the expression of Tn. Here, we introduce a method named EXoO-Tn for large-scale mapping of Tn-glycosylated proteins and glycosylation sites. EXoO-Tn utilizes solid-phase immobilization of proteolytic peptides of proteins, which modifies Tn by glycosyltransferase C1GalT1 with isotopically labeled UDP-Gal( 13 C 6 ), to tag and convert Tn to Gal( 13 C 6 )-Tn, which gives rise to a unique glycan mass. The exquisite Gal( 13 C 6 ) modified Tn are then recognized by a human-gut-bacterial enzyme, OpeRATOR, and released at the N-termini of the Gal( 13 C 6 )-Tn-occupied Ser/Thr residues from immobilized peptides to yield site-containing glycopeptides. The effectiveness of EXoO-Tn was benchmarked by analyzing Jurkat cells, where 947 Tn-glycosylation sites from 480 glycoproteins were mapped. The EXoO-Tn was further applied to the analysis of pancreatic cancer sera, where Tn-glycoproteins were identified. Given the significance of Tn in cancer, EXoO-Tn is anticipated to have broad translational and clinical utilities.

Glycosylation is a major post-translational modification of proteins that regulates many biological processes including protein folding, structure stability, receptor activation, and immune responses. The glycans attached to proteins represent an important determinant of the protein interaction-specificity and maintain the 3D structure of proteins. Mass spectrometry (MS) is one of the most efficient tools used in the current studies of glycoproteins and structure of their glycoforms. Collision energy (CE) is a crucial instrument parameter that can be exploited to improve structural resolution because different linkages of glycan units show different stabilities under CID/HCD fragmentation. Here we report the utility of CE modulation for qualitative and quantitative analysis of site- and structure-specific glycoforms of proteins. Using CE modulation, we were able to break selectively specific glycan linkages on intact glycopeptides and get, to some degree, structure-specific mass spectrometric signals. Structure- and CE-specific oxonium ions provide sufficient information for the resolution of outer arm structure motifs with recognized biological functions. The complementary Y-ions, generated under optimized low CE (soft) conditions, provide additional structural information including features specific to the chitobiose core. This methodology of multiple CE fragmentation without merging spectral information can significantly improve confidence of glycopeptide identification and structural resolution by providing additional information to the established glycopeptide-search algorithms and tools.

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Protein N-glycosylation is ubiquitous in the brain and is closely related to cognition and memory. Alzheimer's disease (AD) is a multifactorial disorder that lacks a clear pathogenesis and treatment. Aberrant N-glycosylation has been suggested to be involved in AD pathology. However, the systematic variations in protein N-glycosylation and their roles in AD have not been thoroughly investigated due to technical challenges. Here, we applied multilayered N-glycoproteomics to quantify the global protein expression levels, N-glycosylation sites, N-glycans, and site-specific N-glycopeptides in AD (APP/PS1 transgenic) and wild-type mouse brains. The N-glycoproteomic landscape exhibited highly complex site-specific heterogeneity in AD mouse brains. The generally dysregulated N-glycosylation in AD, which involved proteins such as glutamate receptors as well as fucosylated and oligomannose glycans, were explored by quantitative analyses. Furthermore, functional studies revealed the crucial effects of N-glycosylation on proteins and neurons. Our work provides a systematic multilayered N-glycoproteomic strategy for AD and can be applied to diverse biological systems.

Mannose-6-phosphate (M6P) glycosylation is an important post-translational modification (PTM) and plays a crucial role in transferring lysosomal hydrolases to lysosome, and is involved in several other biological processes. Aberrant M6P modifications have been implicated in lysosomal storage diseases and numerous other disorders including Alzheimer's disease and cancer. Research on profiling of intact M6P glycopeptides remains challenging due to its extremely low stoichiometry. Here we propose a dual-mode affinity approach to enrich M6P glycopeptides by dual-functional titanium(IV) immobilized metal affinity chromatography [Ti(IV)-IMAC] materials. In combination with state-of-the-art mass spectrometry and database search engine, we profiled 237 intact M6P glycopeptides corresponding to 81 M6P glycoproteins in five types of tissues in mouse, representing the first large-scale profiling of M6P glycosylation in mouse samples. The analysis of M6P glycoforms revealed the predominant glycan substrates of this PTM. Gene ontology analysis showed that overrepresented M6P glycoproteins were lysosomal-associated proteins. However, there were still substantial M6P glycoproteins that possessed different subcellular locations and molecular functions. Deep mining of their roles implicated in lysosomal and nonlysosomal function can provide new insights into functional roles of this important yet poorly studied modification.

We report a liquid chromatography coupled to tandem mass spectrometry O-glycoproteomics strategy using data-independent acquisition (DIA) mode for direct analysis of O-glycoproteins. This approach enables characterization of glycopeptides and structures of O-glycans on a proteome-wide scale with quantification of stoichiometries (though it does not allow for direct unambiguous glycosite identification). The method relies on a spectral library of O-glycopeptides; the Glyco-DIA library contains sublibraries obtained from human cell lines and human serum, and it currently covers 2,076 O-glycoproteins (11,452 unique glycopeptide sequences) and the 5 most common core1 O-glycan structures. Applying the Glyco-DIA library to human serum without enrichment for glycopeptides enabled us to identify and quantify 269 distinct glycopeptide sequences bearing up to 5 different core1 O-glycans from 159 glycoproteins in a SingleShot analysis.

Glycosylation, as a biologically important protein post-translational modification, often alters on both glycosites and glycans, simultaneously. However, most of current approaches focused on biased profiling of either glycosites or glycans, and limited by time-consuming process and milligrams of starting protein material. We describe here a simple and integrated spintip-based glycoproteomics technology (termed Glyco-SISPROT) for achieving a comprehensive view of glycoproteome with shorter sample processing time and low microgram starting material. By carefully integrating and optimizing SCX, C18 and Concanavalin A (Con A) packing material and their combination in spintip format, both predigested peptides and protein lysates could be processed by Glyco-SISPROT with high efficiency. More importantly, deglycopeptide, intact glycopeptide and glycans released by multiple glycosidases could be readily collected from the same Glyco-SISPROT workflow for LC-MS analysis. In total, above 1850 glycosites in ˜1770 unique deglycopeptides were characterized from mouse liver by using either 100 μg of predigested peptides or directly using 100 μg of protein lysates, in which about 30% of glycosites were released by both PNGase F and Endos. To the best of our knowledge, this approach should be one of the most comprehensive glycoproteomic approaches by using limited protein starting material. One significant benefit of Glyco-SISPROT is that whole processing time is dramatically reduced from a few days to less than 6 h with good reproducibility when protein lysates were directly processed by Glyco-SISPROT. We expect that this method will be suitable for multi-level glycoproteome analysis of rare biological samples with high sensitivity., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Protein glycosylation is increasingly recognised as an essential requirement for effective microbial infections. Within microbial pathogen's protein glycosylation is used for both defensive and offensive purposes; enabling pathogens to fortify themselves against the host immune response or to disarm the host's ability to resist infection. Although microbial protein glycosylation systems have been recognised for nearly two decades only recently has the true extend of protein glycosylation within microbes begun to be appreciated. A key enabler for this conceptual shift has been the development and application of modern approaches for the characterisation of glycosylation. Over the last decade my research has focused on the development of proteomic tools to probe microbial glycosylation. By developing workflows for glycopeptide enrichment and identification we have demostrated that it is now possible to characterise the glycoproteomes of microbial species in a truely high-throughput manner. Using these high-throughput approaches we have shown a number of bacterial species modify multiple proteins including members of the Campylobacter genus and the pathogens A. baumannii, R. solanacearum and B. cenocepacia. These studies have established that bacterial glycosylation is widespread, that glycan microheterogeneity is common place and that an extensive array of glycans are used to decorate protein compared to Eukaryotic glycosylation systems. Excitingly these approaches developed to characterise O- and N-linked bacterial glycosylation systems are equally amenable to studying newly discovered forms of microbial glycosylation such as Arginine glycosylation as well as glycosylation within the parasitic eukaryotic organisms T. gondii and P. falciparum. This work demonstrates that MS approaches can now be considered an indispensable tool for the elucidation and tracking of microbial glycosylation events.

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., (© 2019 Wiley Periodicals, Inc.)

Hepatocellular carcinoma (HCC) is the third most-common cause of cancer-related death worldwide. Most cases of HCC develop in patients that already have liver cirrhosis and have been recommended for surveillance for an early onset of HCC. Cirrhosis is the final common pathway for several etiologies of liver disease, including hepatitis B and C, alcohol, and increasingly non-alcoholic fatty liver disease. Only 20-30% of patients with HCC are eligible for curative therapy due primarily to inadequate early-detection strategies. Reliable, accurate biomarkers for HCC early detection provide the highest likelihood of curative therapy and survival; however, current early-detection methods that use abdominal ultrasound and serum alpha fetoprotein are inadequate due to poor adherence and limited sensitivity and specificity. There is an urgent need for convenient and highly accurate validated biomarkers for HCC early detection. The theme of this review is the development of new methods to discover glycoprotein-based markers for detection of HCC with mass spectrometry approaches. We outline the non-mass spectrometry based methods that have been used to discover HCC markers including immunoassays, capillary electrophoresis, 2-D gel electrophoresis, and lectin-FLISA assays. We describe the development and results of mass spectrometry-based assays for glycan screening based on either MALDI-MS or ESI analysis. These analyses might be based on the glycan content of serum or on glycan screening for target molecules from serum. We describe some of the specific markers that have been developed as a result, including for proteins such as Haptoglobin, Hemopexin, Kininogen, and others. We discuss the potential role for other technologies, including PGC chromatography and ion mobility, to separate isoforms of glycan markers. Analyses of glycopeptides based on new technologies and innovative softwares are described and also their potential role in discovery of markers of HCC. These technologies include new fragmentation methods such as EThcD and stepped HCD, which can identify large numbers of glycopeptide structures from serum. The key role of lectin extraction in various assays for intact glycopeptides or their truncated versions is also described, where various core-fucosylated and hyperfucosylated glycopeptides have been identified as potential markers of HCC. Finally, we describe the role of LC-MRMs or lectin-FLISA MRMs as a means to validate these glycoprotein markers from patient samples. These technological advancements in mass spectrometry have the potential to lead to novel biomarkers to improve the early detection of HCC., (© 2018 Wiley Periodicals, Inc.)

A relatively novel activation technique, electron-transfer/higher-energy collision dissociation (EThcD) was used in the LC-MS/MS analysis of tryptic glycopeptides enriched with wheat germ agglutinin from human urine samples. We focused on the characterization of mucin-type O-glycopeptides. EThcD in a single spectrum provided information on both the peptide modified and the glycan carried. Unexpectedly, glycan oxonium ions indicated the presence of O-acetyl, and even O-diacetyl-sialic acids. B and Y fragment ions revealed that (i) in core 1 structures the Gal residue featured the O-acetyl-sialic acid, when there was only one in the glycan; (ii) several glycopeptides featured core 1 glycans with disialic acids, in certain instances O-acetylated; (iii) the disialic acid was linked to the GalNAc residue whatever the degree of O-acetylation; (iv) core 2 isomers with a single O-acetyl-sialic acid were chromatographically resolved. Glycan fragmentation also helped to decipher additional core 2 oligosaccharides: a LacdiNAc-like structure, glycans carrying sialyl Lewis X/A at different stages of O-acetylation, and blood antigens. A sialo core 3 structure was also identified. We believe this is the first study when such structures were characterized from a very complex mixture and were linked not only to a specific protein, but also the sites of modifications have been determined.

The Chinese hamster ovary (CHO) cell line is a major expression system for the production of therapeutic proteins, the majority of which are glycoproteins, such as antibodies and erythropoietin (EPO). The characterization glycosylation profile of therapeutic proteins produced from engineered CHO cells and therapeutic functions, as well as side effects, are critical to understand the important roles of glycosylation. In this study, a large scale glycoproteomic workflow was established and applied to CHO-K1 cells expressing EPO. The workflow includes enrichment of intact glycopeptides from CHO-K1 cell lysate and medium using hydrophilic enrichment, fractionation of the obtained intact glycopeptides (IGPs) by basic reversed phase liquid chromatography (bRPLC), analyzing the glycopeptides using LC-MS/MS, and annotating the results by GPQuest 2.0. A total of 10 338 N-linked glycosite-containing IGPs were identified, representing 1162 unique glycosites in 530 glycoproteins, including 71 unique atypical N-linked IGPs on 18 atypical N-glycosylation sequons with an overrepresentation of the N-X-C motifs. Moreover, we compared the glycoproteins from CHO cell lysate with those from medium using the in-depth N-linked glycoproteome data. The obtained large scale glycoproteomic data from intact N-linked glycopeptides in this study is complementary to the genomic, proteomic, and N-linked glycomic data previously reported for CHO cells. Our method has the potential to monitor the production of recombinant therapeutic glycoproteins.

Protein glycosylation, an important PTM, plays an essential role in a wide range of biological processes such as immune response, intercellular signaling, inflammation, and host-pathogen interaction. Aberrant glycosylation has been correlated with various diseases. However, studying protein glycosylation remains challenging because of low abundance, microheterogeneities of glycosylation sites, and poor ionization efficiency of glycopeptides. Therefore, the development of sensitive and accurate approaches to characterize protein glycosylation is crucial. The identification and characterization of protein glycosylation by MS is referred to as the field of glycoproteomics. Methods such as enrichment, metabolic labeling, and derivatization of glycopeptides in conjunction with different MS techniques and bioinformatics tools, have been developed to achieve an unequivocal quantitative and qualitative characterization of glycoproteins. This review summarizes the recent developments in the field of glycoproteomics over the past 6 years (2012 to 2018)., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The unambiguous mass spectrometric identification and characterization of glycopeptides is crucial to elucidate the micro- and macroheterogeneity of glycoproteins. Here, combining lower and stepped collisional energy fragmentation for the in-depth and site-specific analysis of N- and O-glycopeptides is proposed. Using a set of four representative and biopharmaceutically relevant glycoproteins (IgG, fibrinogen, lactotransferrin, and ribonuclease B), the benefits and limitations of the developed workflow are highlighted and a state-of-the-art blueprint for conducting high-quality in-depth N- and O-glycoproteomic analyses is provided. Further, a modified and improved version of cotton hydrophilic interaction liquid chromatography-based solid phase extraction for glycopeptide enrichment is described. For the unambiguous identification of N-glycopeptides, the use of a conserved yet, rarely employed-fragmentation signature [M peptide +H+ 0,2 X GlcNAc] + is proposed. It is shown for the first time that this fragmentation signature can consistently be found across all N-glycopeptides, but not on O-glycopeptides. Moreover, the use of the relative abundance of oxonium ions to retrieve glycan structure information, for example, differentiation of hybrid- and high-mannose-type N-glycans or differentiation between antenna GlcNAc and bisecting GlcNAc, is systematically and comprehensively evaluated. The findings may increase confidence and comprehensiveness in manual and software-assisted glycoproteomics., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Human serum is a complex body fluid that contains various N-linked and O-linked glycoproteins. Compared with N-linked glycoproteins, the serum O-linked glycoproteins are not well-studied due to their high heterogeneity and their low abundance. Herein, we presented a novel chemoenzymatic method to analyze core-1 type of O-GalNAcylation in human serum. In this approach, the tryptic digest of serum was first subjected to PNGase F treatment to release the N-glycan and was then treated with strong acid to release sialic acid residues from mucin-type O-glycans. In this way, the internal Gal/GalNAc residues were exposed and were oxidized by the galactose oxidase to carry the aldehyde groups. The oxidized O-GalNAcylated peptides were then captured by hydrazide beads and eluted with methoxylamine for LC-MS/MS analysis. The de-N-deglycosylation decreased the abundance of N-glycopeptides, the desialylation simplified the O-glycans and the enzymatic oxidization conferred the enrichment specificity. We have demonstrated that this method was fitted to analyze O-GalNAcylated peptides with high confidence. This method was applied to analyze human serum, which resulted in the identification of 59 O-GalNAc modified peptide sequences corresponding to 38 glycoproteins from 50 μL of serum. This method is expected to have broad applications in the analysis of O-glycoproteome.

Sialic acids are a family of structurally unique and negatively charged nine-carbon sugars, normally found at the terminal positions of glycan chains on glycoproteins and glycolipids. The glycosylation of proteins is a universal post-translational modification in eukaryotic species and regulates essential biological functions, in which the most common sialic acid is N-acetyl-neuraminic acid (2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-onic acid) (Neu5NAc). Because of the properties of sialic acids under general mass spectrometry (MS) conditions, such as instability, ionization discrimination, and mixed adducts, the use of MS in the analysis of protein sialoglycosylation is still challenging. The present review is focused on the application of MS related methodologies to the study of both N- and O-linked sialoglycans. We reviewed MS-based strategies for characterizing sialylation by analyzing intact glycoproteins, proteolytic digested glycopeptides, and released glycans. The review concludes with future perspectives in the field., (© 2017 Wiley Periodicals, Inc.)

SugarQb (www.imba.oeaw.ac.at/sugarqb) is a freely available collection of computational tools for the automated identification of intact glycopeptides from high-resolution HCD MS/MS datasets in the Proteome Discoverer environment. We report the migration of SugarQb to the latest and free version of Proteome Discoverer 2.1, and apply it to the analysis of PNGase F-resistant N-glycopeptides from mouse embryonic stem cells. The analysis of intact glycopeptides highlights unexpected technical limitations to PNGase F-dependent glycoproteomic workflows at the proteome level, and warrants a critical reinterpretation of seminal datasets in the context of N-glycosylation-site prediction., (© 2018 The Authors. Proteomics Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

To elucidate the relationship between the protein function and the diversity and heterogeneity of glycans conjugated to the protein, glycosylation sites, glycan variation, and glycan proportions at each site of the glycoprotein must be analyzed. Glycopeptide-based structural analysis technology using mass spectrometry has been developed; however, complicated analyses of complex spectra obtained by multistage fragmentation are necessary, and sensitivity and throughput of the analyses are low. Therefore, we developed a liquid chromatography/mass spectrometry (MS)-based glycopeptide analysis method to reveal the site-specific glycome (Glycan heterogeneity-based Relational IDentification of Glycopeptide signals on Elution profile, Glyco-RIDGE). This method used accurate masses and retention times of glycopeptides, without requiring MS2, and could be applied to complex mixtures. To increase the number of identified peptide, fractionation of sample glycopeptides for reduction of sample complexity is required. Therefore, in this study, glycopeptides were fractionated into four fractions by hydrophilic interaction chromatography, and each fraction was analyzed using the Glyco-RIDGE method. As a result, many glycopeptides having long glycans were enriched in the highest hydrophilic fraction. Based on the monosaccharide composition, these glycans were thought to be poly-N-acetyllactosamine (polylactosamine [pLN]), and 31 pLN-carrier proteins were identified in HL-60 cells. Gene ontology enrichment analysis revealed that pLN carriers included many molecules related to signal transduction, receptors, and cell adhesion. Thus, these findings provided important insights into the analysis of the glycoproteome using our novel Glyco-RIDGE method. Graphical Abstract ᅟ.

The system-wide site-specific analysis of intact glycopeptides is crucial for understanding the exact functional relevance of protein glycosylation. A dedicated workflow with the capability to simultaneously characterize and quantify intact glycopeptides in a site-specific and high-throughput manner is essential to reveal specific glycosylation alteration patterns in complex biological systems. In this study, an enhanced, dedicated, large-scale site-specific quantitative N-glycoproteomics workflow has been established, which includes improved specific extraction of membrane-bound glycoproteins using the filter aided sample preparation (FASP) method, enhanced enrichment of N-glycopeptides using sequential hydrophilic interaction liquid chromatography (HILIC) and multi-lectin affinity (MLA) enrichment, site-specific N-glycopeptide characterization enabled by EThcD, relative quantitation utilizing isobaric N,N-dimethyl leucine (DiLeu) tags and automated FDR-based large-scale data analysis by Byonic. For the first time, our study shows that HILIC complements to a very large extent to MLA enrichment with only 20% overlapping in enriching intact N-glycopeptides. When applying the developed workflow to site-specific N-glycoproteome study in PANC1 cells, we were able to identify 1067 intact N-glycopeptides, representing 311 glycosylation sites and 88 glycan compositions from 205 glycoproteins. We further applied this approach to study the glycosylation alterations in PKM2 knockout cells vs. parental breast cancer cells and revealed altered N-glycoprotein/N-glycopeptide patterns and very different glycosylation microheterogeneity for different types of glycans. To obtain a more comprehensive map of glycoprotein alterations, N-glycopeptides after treatment with PNGase F were also analyzed. A total of 484 deglycosylated peptides were quantified, among which 81 deglycosylated peptides from 70 glycoproteins showed significant changes. KEGG pathway analysis revealed that the PI3K/Akt signaling pathway was highly enriched, which provided evidence to support the previous finding that PKM2 knockdown cancer cells rely on activation of Akt for their survival. With glycosylation being one of the most important signaling modulators, our results provide additional evidence that signaling pathways are closely regulated by metabolism.

Hybrid mass spectrometry (MS) is an emerging technique for characterizing glycoproteins, which typically display pronounced microheterogeneity. Since hybrid MS combines information from different experimental levels, it crucially depends on computational methods. Here, we describe a novel software tool, MoFi, which integrates hybrid MS data to assign glycans and other post-translational modifications (PTMs) in deconvoluted mass spectra of intact proteins. Its two-stage search algorithm first assigns monosaccharide/PTM compositions to each peak and then compiles a hierarchical list of glycan combinations compatible with these compositions. Importantly, the program only includes those combinations which are supported by a glycan library as derived from glycopeptide or released glycan analysis. By applying MoFi to mass spectra of rituximab, ado-trastuzumab emtansine, and recombinant human erythropoietin, we demonstrate how integration of bottom-up data may be used to refine information collected at the intact protein level. Accordingly, our software reveals that a single mass frequently can be explained by a considerable number of glycoforms. Yet, it simultaneously ranks proteoforms according to their probability, based on a score which is calculated from relative glycan abundances. Notably, glycoforms that comprise identical glycans may nevertheless differ in score if those glycans occupy different sites. Hence, MoFi exposes different layers of complexity that are present in the annotation of a glycoprotein mass spectrum.

Due to the critical role glycation plays in many serious pathological conditions, such as diabetes, it is of great significance to discover protein glycation at an early stage for precaution and prediction of the disease. Here, a method of reductive amination combining dimethylation (RAD) was developed for the quantification of early-stage glycated proteins. The quantitative analysis was first carried out by reducing the samples using NaBH 3 CN or NaBD 3 CN, resulting in a 1 Da mass shift and the stabilization of early-stage protein glycation. The two samples were then digested and isotopically dimethylated to achieve the mass shift of 4 m + 3 n ( m represents the number of N-termini and Lys residues, and n represents the number of glycated sites) between light- and heavy-labeled glycated peptides for quantification. Consequently, the false positive result can be removed according to the different mass shifts of glycated peptides and non-glycated peptides. In quantification of glycated myoglobin, RAD showed good linearity ( R 2 > 0.99) and reproducibility (CVs ≤ 1.6%) in 2 orders of magnitude (1:10-10:1). RAD was then applied to quantify the endogenous glycated proteins in the serum of diabetic patients, revealing significant differences in the glycation level between the patients with complicated retinal detachment and those without. In conclusion, RAD is an effective method for quantifying endogenous glycated proteins.