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Lectin affinity chromatography is one of powerful tools for the study of protein glycosylation. Different lectin proteins can recognize different structures of monosaccharides or oligosaccharide units, allowing for the selective separation of glycopeptides or glycoproteins containing different polysaccharide structures. However, the N-glycans were only partially captured by most of common lectins, reducing the coverage rate of identifying N-glycoconjugates. Recently, it has been reported that the engineering variant of glycan binding protein Fbs1 has a high affinity for innermost Man 3 GlcNAc 2 structure and is able to bind diverse types of N-glycans, which can be suitable for the analysis of protein N-glycosylation. However, efficient immobilization of protein to separation matrix is particularly challenging as it requires the functionality and integrity of the protein to be preserved. Herein, we describe a simple and robust strategy for oriental covalent immobilization of proteins on magnetic nanoparticles by N-terminal selective labeling techniques. We inserted the enterokinase cleavage site to produce the specific N- terminal glycine of protein. Under physiological conditions, the protein was immobilized on the surface of the MNPs by this glycine tag, and the enrichment process could be completed within 30 min. A whole enrichment and purification of glycan and glycopeptides were completed and analyzed by MALDI TOF-MS. The functional materials achieved stable enrichment of glycan structure in different enzyme digestion systems or complex samples, showing excellent anti-interference and applicability., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: The need for stationary phases with unique selectivity in reversed-phase liquid chromatography has been of utmost importance to chromatographers for advancing the analysis of complex samples. Macrocyclic glycopeptide based stationary phases have been widely used for chiral separations with different chromatographic modes such as normal phase, reversed phase, and supercritical fluid chromatography. Given the multimodal retention mechanisms namely π-π complex interaction, hydrogen bonding, dipole-dipole interaction, and strong Coulombic interactions by which analytes are separated using the macrocyclic glycopeptides, these stationary phases are expected to provide novel selectivity when used under the reversed phase for achiral separations., Results: Herein, for the first time we have conducted a systematic study using the improved hydrophobic subtraction model (HSM) which incoporates dipole-dipole interactions to demonstrate the novel selectivity offered by four different macrocyclic glycopeptide based stationary phases, namely NicoShell, TeicoShell, TagShell, and VancoShell. A comparison of the HSM parameters for these columns has been made with 551 commercially available reversed phase stationary phases and the differences in the values point to the importance of adding these columns to the already existing arsenal. These stationary phases offer separations over a wide range of pH and show variability in selectivity depending on the pH of the mobile phase which make them versatile for method development in the reversed phase mode. Additionally, we have provided an actual example of a separation from an Amgen discovery project using the VancoShell column aided by computer-assisted modelling., Significance: This is the first report characterizing macrocyclic glycopeptides for achiral RPLC applications. The selectivity of these stationary phases were found to be unique when compared to other commercially available stationary phases thereby acting as their own class of columns. The unusual selectivity of the columns enabled separation of complex pharmaceutical samples., Competing Interests: Declaration of competing interest DWA is president of AZYP, LLC. All other authors declare no competing financial or personal interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Protein glycosylation acts as a crucial role in regulating protein function and maintaining cellular homeostasis. Efficient peptide enrichment can be utilized to effectively solve the inherent challenges of protein glycosylation analysis to search unknown cancer biomarkers. In this research, a low dimensional porous hydrophilic nanosheets with a multi-level porous structure (Co-MOF-SiO 2 @HA) was synthetized via an easy one-pot method for the efficient enrichment of the N-glycopeptides in the digests of complex biosamples. The synthetized nanosheets Co-MOF-SiO 2 @HA demonstrated excellent enriching performances including a high enrichment capacity (300 mg g -1 calculated), a spectacular selectivity (IgG digests and BSA digests at the molar ratio of 1/1200), and an excellent spatial confinement ability (IgG digests, IgG and BSA at the molar ratio of 1/1000/1000). As an explore result, after the enrichment of human colorectal cancer tissue and human healthy tissue by the nanosheets, several proteins related to cancers and one protein directly related to well-known human colorectal cancer were identified by detecting the corresponding glycopeptides. It presented the potential value of the feasibility of this analysis mode by nanosheets Co-MOF-SiO 2 @HA in proteomic analysis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Protein glycosylation, one of the most important biologically relevant post-translational modifications for biomarker discovery, faces analytical challenges due to heterogeneous glycosite, diverse glycans, and mass spectrometry limitations. Glycopeptide enrichment by removing abundant hydrophobic peptides helps overcome some of these obstacles. Hydrophilic interaction liquid chromatography (HILIC), known for its selectivity, glycan separations, intact glycopeptide enrichment, and compatibility with mass spectrometry, has seen recent advancements in stationary phases like Amide-80, glycoHILIC, amino acids or peptides for improved HILIC-based glycopeptide analysis. Utilization of these materials can improve glycopeptide enrichment through solid-phase extraction and separation via high-performance liquid chromatography. Additionally, using glycopeptides themselves to modify HILIC stationary phases holds promise for improving selectivity and sensitivity in glycosylation analysis. Additionally, HILIC has capability to assess the information about glycosites and structural information of glycans. This review summarizes recent breakthroughs in HILIC stationary materials, highlighting their impact on glycopeptide analysis. Ongoing research on advanced materials continues to refine HILIC's performance, solidifying its value as a tool for exploring protein glycosylation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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.)

Bacterial pathogens can cause a broad range of infections with detrimental effects on health. Vaccine development is essential as multi-drug resistance in bacterial infections is a rising concern. Recombinantly produced proteins carrying O-antigen glycosylation are promising glycoconjugate vaccine candidates to prevent bacterial infections. However, methods for their comprehensive structural characterization are lacking. Here, we present a bottom-up approach for their site-specific characterization, detecting N-glycopeptides by nano reversed-phase liquid chromatography-mass spectrometry (RP-LC-MS). Glycopeptide analyses revealed information on partial site-occupancy and site-specific glycosylation heterogeneity and helped corroborate the polysaccharide structures and their modifications. Bottom-up analysis was complemented by intact glycoprotein analysis using nano RP-LC-MS allowing the fast visualization of the polysaccharide distribution in the intact glycoconjugate. At the glycopeptide level, the model glycoconjugates analyzed showed different repeat unit (RU) distributions that spanned from 1 to 21 RUs attached to each of the different glycosylation sites. Interestingly, the intact glycoprotein analysis displayed a RU distribution ranging from 1 to 28 RUs, showing the predominant species when the different glycopeptide distributions are combined in the intact glycoconjugate. The complete workflow based on LC-MS measurements allows detailed and comprehensive analysis of the glycosylation state of glycoconjugate vaccines., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: A.B.M., R.D., V.D., A.A.K., J.G., M.B., and C.A. are employees of Janssen Vaccines. All other authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.)

Precision mapping of site-specific glycans using mass spectrometry is vital in glycoproteomics. However, the diversity of glycan compositions across species often exceeds database capacity, hindering the identification of rare glycans. Here, we introduce pGlycoNovo, a software within the pGlyco3 software environment, which employs a glycan first-based full-range Y-ion dynamic searching strategy. pGlycoNovo enables de novo identification of intact glycopeptides with rare glycans by considering all possible monosaccharide combinations, expanding the glycan search space to 16~1000 times compared to non-open search methods, while maintaining accuracy, sensitivity and speed. Reanalysis of SARS Covid-2 spike protein glycosylation data revealed 230 additional site-specific N-glycans and 30 previously unreported O-glycans. pGlycoNovo demonstrated high complementarity to six other tools and superior search speed. It enables characterization of site-specific N-glycosylation across five evolutionarily distant species, contributing to a dataset of 32,549 site-specific glycans on 4602 proteins, including 2409 site-specific rare glycans, and uncovering unexpected glycan fragments., (© 2024. The Author(s).)

High-throughput intact glycopeptide analysis is crucial for elucidating the physiological and pathological status of the glycans attached to each glycoprotein. Mass spectrometry-based glycoproteomic methods are challenging because of the diversity and heterogeneity of glycan structures. Therefore, we developed an MS1-based site-specific glycoform analysis method named "Glycan heterogeneity-based Relational IDentification of Glycopeptide signals on Elution profile (Glyco-RIDGE)" for a more comprehensive analysis. This method detects glycopeptide signals as a cluster based on the mass and chromatographic properties of glycopeptides and then searches for each combination of core peptides and glycan compositions by matching their mass and retention time differences. Here, we developed a novel browser-based software named GRable for semi-automated Glyco-RIDGE analysis with significant improvements in glycopeptide detection algorithms, including "parallel clustering." This unique function improved the comprehensiveness of glycopeptide detection and allowed the analysis to focus on specific glycan structures, such as pauci-mannose. The other notable improvement is evaluating the "confidence level" of the GRable results, especially using MS2 information. This function facilitated reduced misassignment of the core peptide and glycan composition and improved the interpretation of the results. Additional improved points of the algorithms are "correction function" for accurate monoisotopic peak picking; one-to-one correspondence of clusters and core peptides even for multiply sialylated glycopeptides; and "inter-cluster analysis" function for understanding the reason for detected but unmatched clusters. The significance of these improvements was demonstrated using purified and crude glycoprotein samples, showing that GRable allowed site-specific glycoform analysis of intact sialylated glycoproteins on a large-scale and in-depth. Therefore, this software will help us analyze the status and changes in glycans to obtain biological and clinical insights into protein glycosylation by complementing the comprehensiveness of MS2-based glycoproteomics. GRable can be freely run online using a web browser via the GlyCosmos Portal (https://glycosmos.org/grable)., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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.)

Alterations in glycoprotein abundance and glycan structures are closely linked to numerous diseases. The quantitative exploration of glycoproteomics is pivotal for biomarker discovery, but comprehensive analysis within biological samples remains challenging due to low abundance, complexity, and lack of universal technology. We developed a multiplex glycoproteomic approach using an LC-ESI-MS platform for direct comparison of glycoproteomic quantitation. Glycopeptides were isotopically labeled during cell culture, achieving high labeling efficiency (≥ 95 %) for both glycans and peptides. Quantitation was validated by mixing the same cell line in a 1:1:1:1 ratio, with mathematical correction applied to deconvolute the ratios. This method proved reliable and was applied to a comparative glycoproteomic study of three breast cancer cell lines (HTB22, MDA-MB-231, MDA-MB-231BR) and one brain cancer cell line (CRL-1620), quantifying glycopeptides from three replicates. The expression of glycopeptides was relatively quantified, and up/down-regulation between cell lines was investigated. This approach provided insights into glycosylation microheterogeneity, crucial for breast cancer brain metastasis research. Benefits include eliminating fluctuations from nano electrospray ionization and reducing analysis time, enabling up to 4-plex profiling in a single injection. Metabolic labeling introduced mass differences at the MS1 level, ensuring increased sensitivity and higher resolution for accurate quantitation. SIGNIFICANCE: Alternations in glycoprotein abundance, changes in glycosylation levels, and variations in glycan structures are closely linked to numerous diseases. The quantitative exploration of glycoproteomics has emerged as a popular area of research for biomarker discovery. However, conducting a comprehensive quantitative analysis of the glycoproteome within biological samples remains challenging due to low abundance, inherent complexities, and the absence of universal quantitative technology. Here, we developed a multiplex glycoproteomic approach using an LC-ESI-MS platform to facilitate direct comparison of glycoproteomic quantitation and enhance throughput. This approach offers benefits such as eliminating quantitative fluctuations arising from nano electrospray ionization (ESI) and reducing analysis time, enabling up to 4-plex glycoproteomic profiling in a single injection. Glycopeptides were stable isotopic labeled during cell culture procedure, attaching to monosaccharides, amino acids, or both. We achieved a high labeling efficiency (≥ 95 %) for both glycans and peptides. Quantitation validation was tested on glycopeptides by mixing the same cell line with 1:1:1:1 ratio. Due to the overlapped isotopes, a mathematical correction was applied to deconvolute the ratio of 4-plex glycopeptides. This method demonstrated quantitative reliability and was successfully applied to a comparative glycoproteomic study of three breast cancer cells (HTB22, MDA-MB-231, and MDA-MB-231BR) and one brain cancer cell (CRL-1620), identifying a total of 264 glycopeptides from three replicates. The expression of glycopeptides among these four cells was relatively quantified and up/down-regulation between two cell lines was investigated. The exploration of glycosylation microheterogeneity through glycopeptide quantification may offer valuable insights for further investigation into breast cancer brain metastasis. Conclusion: The primary advantage of our presented work lies in the multiplexing offered by combining two established labeling techniques, SILAC and IDAWG, both of which have been effectively used and widely cited in the scientific community. This combination enhances the applicability and accuracy of our method, as demonstrated by the extensive citations and successful use of these techniques independently. We believe that this multiplexing approach significantly advances the field, despite the method's current limitation to cell systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Posttranslational modifications (PTMs) of proteins regulate several biological processes, and investigating their diversity is crucial for understanding the mechanisms of cell regulation. Glycosylation is one of the most complex posttranslational modifications that control fundamental cellular processes such as protein folding, protein trafficking, host-pathogen interactions, cell adhesion, and cytokine receptor signaling networks. N-linked glycosylation denotes the attachment of glycans (oligosaccharides) to a nitrogen atom of asparagine (N) residues in the consensus motif Asn-X-Ser/Thr (NXS/T), where X is any amino acid except proline. Therefore, mutations in this posttranslational modification (i.e., N-glycosylation) site cause many human genetic diseases, including cancer. In the past decade, high-throughput quantitative proteome profiling tools have significantly renewed our interest in discovering novel cancer diagnostic or prognostic biomarkers through the simultaneous examination of the enormous amount of high-quality data of thousands of proteins and genes in complex biological systems. In this chapter, we describe how aberrant N-linked glycopeptides could be selectively identified as novel single tumor markers through the use of mass spectrometry (MS)-based proteomics, also known as Solid-phase extraction of N-glycopeptides (SPEG), and reasonable hypotheses that have the potential capacity to revolutionize biomarker discovery and bring those markers to the clinic as early as possible., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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.)

Highly purified human menopausal gonadotropin (HP-hMG [Menopur ® , Ferring Pharmaceuticals, Saint-Prex, Switzerland]) contains a 1:1 ratio of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). This analysis aimed to assess gonadotropin (FSH, LH and hCG) abundance in HP-hMG and clarify the source of hCG by assessing the presence of sulfated glycans, which are diagnostic for pituitary hCG forms due to their distinct glycosylation patterns. Additionally, the purity of each sample, their specific components, and their oxidation levels were assessed. HP-hMG samples (three of Menopur ® and two of Menogon ® Ferring Pharmaceuticals, Saint-Prex, Switzerland) were included in the current analyses. Brevactid ® (urinary hCG; Ferring Pharmaceuticals, Saint-Prex, Switzerland) and Ovidrel ® (recombinant hCG; Merck KGaA, Darmstadt, Germany) were used as control samples. Glycopeptide mapping and analysis of impurities were carried out by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Oxidation was assessed through reducing peptide mapping using LC-MS/MS. The FSH and LH in the HP-hMG samples showed sulfated glycans, while no signals of sulfated glycopeptides were detected on any site of the beta subunit of hCG. HP-hMG test samples presented the same hCG glycan distribution as the control sample (placental hCG, Brevactid ® ) extracted from the urine of pregnant women, suggesting a non-pituitary source of hCG. Protein impurities were estimated to constitute approximately 20-30% of the entire HP-hMG protein content in the test samples. More than 200 non-gonadotropin proteins were identified in the HP-hMG test samples, of which several were involved in embryonic development or pregnancy. The alpha subunit of the tested samples was strongly oxidized, with a relative abundance of 20% of the total gonadotropin content. Without taking into account all the protein impurities, the beta subunit of LH was detected only in traces (0.9-1.2%) in all tested HP-HMG samples, confirming the data obtained by intact molecule analysis, while high levels of beta hCG (18-47%) were observed. Advanced molecular analysis of HP-hMG indicates a primarily placental origin of hCG, as evidenced by the absence of hCG sulfated glycans and the predominance of placental non-sulfated hCG in LH activity. The analysis revealed 20-30% of protein impurities and a significant presence of oxidized forms in the HP-hMG samples. These findings are critical for understanding the quality, safety, and clinical profile of HP-hMG.

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.

Aberrant glycosylation has gained significant interest for biomarker discovery. However, low detectability, complex glycan structures, and heterogeneity present challenges in glycoprotein assay development. Using haptoglobin (Hp) as a model, we developed an integrated platform combining functionalized magnetic nanoparticles and zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) for highly specific glycopeptide enrichment, followed by a data-independent acquisition (DIA) strategy to establish a deep cancer-specific Hp-glycosylation profile in hepatitis B virus (HBV, n = 5) and hepatocellular carcinoma (HCC, n = 5) patients. The DIA strategy established one of the deepest Hp-glycosylation landscapes (1029 glycopeptides, 130 glycans) across serum samples, including 54 glycopeptides exclusively detected in HCC patients. Additionally, single-shot DIA searches against a DIA-based spectral library outperformed the DDA approach by 2-3-fold glycopeptide coverage across patients. Among the four N-glycan sites on Hp (N-184, N-207, N-211, N-241), the total glycan type distribution revealed significantly enhanced detection of combined fucosylated-sialylated glycans, which were the most dominant glycoforms identified in HCC patients. Quantitation analysis revealed 48 glycopeptides significantly enriched in HCC ( p < 0.05), including a hybrid monosialylated triantennary glycopeptide on the N-184 site with nearly none-to-all elevation to differentiate HCC from the HBV group (HCC/HBV ratio: 2462 ± 766, p < 0.05). In summary, DIA-MS presents an unbiased and comprehensive alternative for targeted glycoproteomics to guide discovery and validation of glyco-biomarkers.

Protein glycosylation plays a crucial role in various biological processes and is related to various diseases. Highly specific enrichment of glycopeptides before mass spectrometry detection is crucial for comprehensive glycoproteomic analysis. However, it still remains a great challenge due to the absence of affinity materials with excellent enrichment efficiency. In this work, a triazine structure linked by a -NH- bond of two-dimensional (2-D) covalent organic framework (COF) nanosheets was synthesized as an affinity adsorbent for the selective capture of glycopeptides. In particular, by introducing hydrophilic monomers via a bottom-up approach, the 2-D COF (denoted as NENP-1) nanosheets were provided with abundant amino groups and inherent hydrophilicity. Owing to the specific surface area and excessive accessible sites for hydrophilicity, the resulting NENP-1 nanosheets exhibited an outstanding glycopeptide enrichment efficiency from standard samples with a superior detection sensitivity (1 × 10 -10 M), good enrichment selectivity (1 : 800, HRP tryptic digest to BSA protein), excellent binding capacity (100 mg g -1 ), great reusability, and recovery (60.2%). Furthermore, using the NENP-1 nanosheet adsorbent, twenty-four endogenous glycopeptides in the serum of patients with gastric cancer were successfully identified by LC-MS/MS technology, which illustrates a promising prospective of hydrophilic COF nanosheets in glycoproteomics research.

N-linked glycoproteins are rich in seminal plasma, playing essential roles in supporting sperm function and fertilization process. The alteration of seminal plasma glycans and its correspond glycoproteins may lead to sperm dysfunction and even infertility. In present study, an integrative analysis of glycoproteomic and proteomic was performed to investigate the changes of site-specific glycans and glycoptoteins in seminal plasma of asthenozoospermia. By large scale profiling and quantifying 5,018 intact N-glycopeptides in seminal plasma, we identified 92 intact N-glycopeptides from 34 glycoproteins changed in asthenozoospermia. Especially, fucosylated glycans containing lewis x, lewis y and core fucosylation were significantly up-regulated in asthenozoospermia compared to healthy donors. The up-regulation of fucosylated glycans in seminal plasma may interfere sperm surface compositions and regulation of immune response, which subsequently disrupts sperm function. Three differentiated expression of seminal vesicle-specific glycoproteins (fibronectin, seminogelin-2, and glycodelin) were also detected with fucosylation alteration in seminal plasma of asthenozoospermia. The interpretation of the altered site-specific glycan structures provides data for the diagnosis and etiology analysis of male infertility, as well as providing new insights into the potential therapeutic targets for male infertility., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

α -Lactalbumin, an abundant protein present in the milk of most mammals, is associated with biological, nutritional and technological functionality. Its sequence presents N-glycosylation motifs, the occupancy of which is species-specific, ranging from no to full occupancy. Here, we investigated the N-glycosylation of bovine α-lactalbumin in colostrum and milk sampled from four individual cows, each at 9 time points starting from the day of calving up to 28.0 d post-partum. Using a glycopeptide-centric mass spectrometry-based glycoproteomics approach, we identified N-glycosylation at both Asn residues found in the canonical Asn-Xxx-Ser/Thr motif, i.e. Asn45 and Asn74 of the secreted protein. We found similar glycan profiles in all four cows, with partial site occupancies, averaging at 35% and 4% for Asn45 and Asn74, respectively. No substantial changes in occupancy occurred over lactation at either site. Fucosylation, sialylation, primarily with N-acetylneuraminic acid (Neu5Ac), and a high ratio of N,N'-diacetyllactosamine (LacdiNAc)/N-acetyllactosamine (LacNAc) motifs were characteristic features of the identified N-glycans. While no substantial changes occurred in site occupancy at either site during lactation, the glycoproteoform (i.e. glycosylated form of the protein) profile revealed dynamic changes; the maturation of the α-lactalbumin glycoproteoform repertoire from colostrum to mature milk was marked by substantial increases in neutral glycans and the number of LacNAc motifs per glycan, at the expense of LacdiNAc motifs. While the implications of α-lactalbumin N-glycosylation on functionality are still unclear, we speculate that N-glycosylation at Asn74 results in a structurally and functionally different protein, due to competition with the formation of its two intra-molecular disulphide bridges., (© The Author(s) 2024. Published by Oxford University Press.)

Accurate glycopeptide identification in mass spectrometry-based glycoproteomics is a challenging problem at scale. Recent innovation has been made in increasing the scope and accuracy of glycopeptide identifications, with more precise uncertainty estimates for each part of the structure. We present a dynamically adapting relative retention time model for detecting and correcting ambiguous glycan assignments that are difficult to detect from fragmentation alone, a layered approach to glycopeptide fragmentation modeling that improves N-glycopeptide identification in samples without compromising identification quality, and a site-specific method to increase the depth of the glycoproteome confidently identifiable even further. We demonstrate our techniques on a set of previously published datasets, showing the performance gains at each stage of optimization. These techniques are provided in the open-source glycomics and glycoproteomics platform GlycReSoft available at https://github.com/mobiusklein/glycresoft ., (© 2024. The Author(s).)

Monoclonal antibodies like Herceptin play a pivotal role in modern therapeutics, with their glycosylation patterns significantly influencing their bioactivity. To characterize the N-glycan profile and their relative abundance in Herceptin, we employed two analytical methods: hydrophilic interaction chromatography with fluorescence detection (HILIC-FLD) for released glycans and liquid chromatography tandem mass spectrometry (LC-MS/MS) for glycopeptides. Our analysis included 21 European Union (EU)-Herceptin lots and 14 United States (US)-Herceptin lots. HILIC-FLD detected 25 glycan species, including positional isomers, revealing comparable chromatographic profiles for both EU and US lots. On the other hand, LC-MS/MS identified 26 glycoforms within the glycopeptide EEQYNSTYR. Both methods showed that a subset of glycans dominated the total abundance. Notably, EU-Herceptin lots with an expiration date of October 2022 exhibited increased levels of afucosylated and high mannose N-glycans. Our statistical comparisons showed that the difference in quantitative results between HILIC-FLD and LC-MS/MS is significant, indicating that the absolute quantitative values depend on the choice of the analytical method. However, despite these differences, both methods demonstrated a strong correlation in relative glycan proportions. This study contributes to the comprehensive analysis of Herceptin's glycosylation, offering insights into the influence of analytical methods on glycan quantification and providing valuable information for the biopharmaceutical industry., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Taking into account the drug resistance of antibiotics, teicoplanin has been banned in the veterinary field. Also, it brings threat to people's health when they eat foods containing teicoplanin residue. In addition, the abuse of teicoplanin in humans and food animals also poses a potential risk to water. Therefore, it is crucial to purify teicoplanin from food before quantifying its amount. In this study, researchers employed boronate affinity-based controlled oriented surface imprinting technique to produce molecularly imprinted polymers (MIPs) for the isolation of teicoplanin. The 3-fluoro-4-formylphenylboronic acid-functionalized silica nanoparticle substrate was first used as the supporting material for immobilizing teicoplanin. Next, the substrate surface was coated with an imprinting coating whose thickness could be controlled, produced through the self-copolymerization of dopamine and m -aminophenylboronic acid (APBA) in water. After the template was removed, 3D cavities that matched the template were created in the imprinting layer. The prepared teicoplanin-imprinted silica nanoparticles exhibited several significant satisfactory results such as good specificity, high binding capacity (46.9 ± 2.3 mg g -1 ), moderate binding constant ((5.46 ± 0.18) × 10 -5 M -1 ), fast kinetics (8 min) and low binding pH (pH 5.0) toward teicoplanin. The teicoplanin-imprinted silica nanoparticles could still be reused after seven cycles of adsorption-desorption, which indicated a high chemical stability. In addition, recoveries of the proposed method for teicoplanin at three spiked levels in milk and water ranged from 91.8 to 105.6% and 92.3 to 97.4%, respectively. The teicoplanin-imprinted silica nanoparticles are capable of identifying the target teicoplanin in real samples in a simple, fast, selective and efficient manner.

Tandem mass spectrometry coupled with liquid chromatography (LC-MS/MS) has proven a versatile tool for the identification and quantification of proteins and their post-translational modifications (PTMs). Protein glycosylation is a critical PTM for the stability and biological function of many proteins, but full characterization of site-specific glycosylation of proteins remains analytically challenging. Collision-induced dissociation (CID) is the most common fragmentation method used in LC-MS/MS workflows, but the loss of labile modifications renders CID inappropriate for detailed characterization of site-specific glycosylation. Electron-based dissociation methods provide alternatives that retain intact glycopeptide fragments for unambiguous site localization, but these methods often underperform CID due to increased reaction times and reduced efficiency. Electron-activated dissociation (EAD) is another strategy for glycopeptide fragmentation. Here, we use a ZenoTOF 7600 SCIEX instrument to compare the performance of various fragmentation techniques for the analysis of a complex mixture of mammalian O - and N -glycopeptides. We found CID fragmentation identified the most glycopeptides and generally produced higher quality spectra, but EAD provided improved confidence in glycosylation site localization. Supplementing EAD with CID fragmentation (EAciD) further increased the number and quality of glycopeptide identifications, while retaining localization confidence. These methods will be useful for glycoproteomics workflows for either optimal glycopeptide identification or characterization.

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.

Alpha-1-acid glycoprotein (AGP) is a heterogeneous glycoprotein fulfilling key roles in many biological processes, including transport of drugs and hormones and modulation of inflammatory and immune responses. The glycoform profile of AGP is known to change depending on (patho)physiological states such as inflammatory diseases or pregnancy. Besides complexity originating from five N-glycosylation sites, the heterogeneity of the AGP further expands to genetic variants. To allow in-depth characterization of this intriguing protein, we developed a method using anion exchange chromatography (AEX) coupled to mass spectrometry (MS) revealing the presence of over 400 proteoforms differing in their glycosylation or genetic variants. More precisely, we could determine that AGP mainly consists of highly sialylated higher antennary structures with on average 16 sialic acids and 0 or 1 fucose per protein. Interestingly, a slightly higher level of fucosylation was observed for AGP1 variants compared to that of AGP2. Proteoform assignment was supported by integrating data from complementary MS-based approaches, including AEX-MS of an exoglycosidase-treated sample and glycopeptide analysis after tryptic digestion. The developed analytical method was applied to characterize AGP from plasma of women during and after pregnancy, revealing differences in glycosylation profiles, specifically in the number of antennae, HexHexNAc units, and sialic acids.

The analysis of the structures of glycans present on glycoproteins is an essential component for determining glycoprotein function; however, detailed glycan structural assignment on glycopeptides from proteomics mass spectrometric data remains challenging. Glycoproteomic analysis by mass spectrometry currently can provide significant, yet incomplete, information about the glycans present, including the glycan monosaccharide composition and in some circumstances the site(s) of glycosylation. Advancements in mass spectrometric resolution, using high-mass accuracy instrumentation and tailored MS/MS fragmentation parameters, coupled with a dedicated definition of diagnostic fragmentation ions have enabled the determination of some glycan structural features, or glycotopes, expressed on glycopeptides. Here we present a collation of diagnostic glycan fragments produced by traditional positive-ion-mode reversed-phase LC-ESI MS/MS proteomic workflows and describe the specific fragmentation energy settings required to identify specific glycotopes presented on N- or O-linked glycopeptides in a typical proteomics MS/MS experiment.

Currently, glycopeptide quantitation is mainly based on relative quantitation due to absolute quantitation requiring isotope-labeled or standard glycopeptides which may not be commercially available or are very costly and time consuming to synthesize. To address this grand challenge, coulometric mass spectrometry (CMS), based on the combination of electrochemistry (EC) and mass spectrometry (MS), was utilized to quantify electrochemically active glycopeptides without the need of using standard materials. In this study, we studied tyrosine-containing glycopeptides, NYIVGQPSS(β-GlcNAc)TGNL-OH and NYSVPSS(β-GlcNAc)TGNL-OH, and successfully quantified them directly with CMS with a discrepancy of less than 5% between the CMS measured amount and the theoretical amount. Taking one step further, we applied this approach to quantify glycopeptides generated from the digestion of NIST mAb, a monoclonal antibody reference material. Through HILIC column separation, five N297 glycopeptides resulting from NIST mAb tryptic digestion were successfully separated and quantified by CMS for an absolute amount without the use of any standard materials. This study indicates the potential utility of CMS for quantitative proteomics research.

Dysregulation of protein core-fucosylation plays a pivotal role in the onset, progression, and immunosuppression of cancer. However, analyzing core-fucosylation, especially the accurate determination of the core-fucosylation (CF) site occupancy ratio, remains challenging. To address these problems, we developed a truncation strategy that efficiently converts intact glycopeptides with hundreds of different glycans into two truncated forms, i.e., a monosaccharide HexNAc and a disaccharide HexNAc+core-fucose. Further combination with data-independent analysis to form an integrated platform allowed the measurement of site-specific core-fucosylation abundances and the determination of the CF occupancy ratio with high reproducibility. Notably, three times CF sites were identified using this strategy compared to conventional methods based on intact glycopeptides. Application of this platform to characterize protein core-fucosylation in two breast cancer cell lines, i.e., MDA-MB-231 and MCF7, yields a total of 1615 unique glycosites and about 900 CF sites from one single LC-MS/MS analysis. Differential analysis unraveled the distinct glycosylation pattern for over 201 cell surface drug targets between breast cancer subtypes and provides insights into developing new therapeutic strategies to aid precision medicine. Given the robust performance of this platform, it would have broad application in discovering novel biomarkers based on the CF glycosylation pattern, investigating cancer mechanisms, as well as detecting new intervention targets.

The study of glycoproteomics presents a set of unique challenges, primarily due to the low abundance of glycopeptides and their intricate heterogeneity, which is specific to each site. Glycoproteins play a crucial role in numerous biological functions, including cell signaling, adhesion, and intercellular communication, and are increasingly recognized as vital markers in the diagnosis and study of various diseases. Consequently, a quantitative approach to glycopeptide research is essential. One effective strategy to address this need is the use of multiplex glycopeptide labeling. By harnessing the synergies of 15 N metabolic labeling via the isotopic detection of amino sugars with glutamine (IDAWG) technique for glycan parts and tandem mass tag (TMT)pro labeling for peptide backbones, we have developed a method that allows for the accurate quantification and comparison of multiple samples simultaneously. The adoption of the liquid chromatography-synchronous precursor selection (LC-SPS-MS3) technique minimizes fragmentation interference, enhancing data reliability, as shown by a 97% TMT labeling efficiency. This method allows for detailed, high-throughput analysis of 32 diverse samples from 231BR cell lines, using both 14 N and 15 N glycopeptides at a 1:1 ratio. A key component of our methodology was the precise correction for isotope and TMTpro distortions, significantly improving quantification accuracy to less than 5% distortion. This breakthrough enhances the efficiency and accuracy of glycoproteomic studies, increasing our understanding of glycoproteins in health and disease. Its applicability to various cancer cell types sets a new standard in quantitative glycoproteomics, enabling deeper investigation into glycopeptide profiles., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

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.

Glycosylation is a common modification across living organisms and plays a central role in understanding biological systems and disease. Our ability to probe the gylcome has grown exponentially in the past several decades. However, further improvements to the analytical toolbox available to researchers would allow for increased capabilities to probe structure and function of biological systems and to improve disease treatment. This article applies the developing technique of two-dimensional Fourier transform ion cyclotron resonance mass spectrometry to a glycoproteomic workflow for the standard glycoproteins coral tree lectin (CTL) and bovine ribonuclease B (BRB) to demonstrate its feasibility as a tool for glycoproteomic workflows. 2D infrared multiphoton dissociation and electron capture dissociation spectra of CTL reveal comparable structural information to their 1D counterparts, confirming the site of glycosylation and monosaccharide composition of the glycan. Spectra collected in 2D of BRB reveal correlation lines of fragment ion scans and vertical precursor ion scans for data collected using infrared multiphoton dissociation and diagonal cleavage lines for data collected by electron capture dissociation. The use of similar techniques for glycoproteomic analysis may prove valuable in instances where chromatographic separation is undesirable or quadrupole isolation is insufficient.

Glycoproteins play important roles in numerous physiological processes and are often implicated in disease. Analysis of site-specific protein glycobiology through glycoproteomics has evolved rapidly in recent years thanks to hardware and software innovations. Particularly, the introduction of parallel accumulation serial fragmentation (PASEF) on hybrid trapped ion mobility time-of-flight mass spectrometry instruments combined deep proteome sequencing with separation of (near-)isobaric precursor ions or converging isotope envelopes through ion mobility separation. However, the reported use of PASEF in integrated glycoproteomics workflows to comprehensively capture the glycoproteome is still limited. To this end, we developed an integrated methodology using timsTOF Pro 2 to enhance N-glycopeptide identifications in complex mixtures. We systematically optimized the ion optics tuning, collision energies, mobility isolation width, and the use of dopant-enriched nitrogen gas (DEN). Thus, we obtained a marked increase in unique glycopeptide identification rates compared to standard proteomics settings, showcasing our results on a large set of glycopeptides. With short liquid chromatography gradients of 30 min, we increased the number of unique N-glycopeptide identifications in human plasma samples from around 100 identifications under standard proteomics conditions to up to 1500 with our optimized glycoproteomics approach, highlighting the need for tailored optimizations to obtain comprehensive data.

Glycosylation, the major post-translational modification of proteins, significantly increases the diversity of proteoforms. Glycans are involved in a variety of pivotal structural and functional roles of proteins, and changes in glycosylation are profoundly connected to the progression of numerous diseases. Mass spectrometry (MS) has emerged as the gold standard for glycan and glycopeptide analysis because of its high sensitivity and the wealth of fragmentation information that can be obtained. Various separation techniques have been employed to resolve glycan and glycopeptide isomers at the front end of the MS. However, differentiating structures of isobaric and isomeric glycopeptides constitutes a challenge in MS-based characterization. Many reports described the use of various ion mobility-mass spectrometry (IM-MS) techniques for glycomic analyses. Nevertheless, very few studies have focused on N- and O-linked site-specific glycopeptidomic analysis. Unlike glycomics, glycoproteomics presents a multitude of inherent challenges in microheterogeneity, which are further exacerbated by the lack of dedicated bioinformatics tools. In this review, we cover recent advances made towards the growing field of site-specific glycosylation analysis using IM-MS with a specific emphasis on the MS techniques and capabilities in resolving isomeric peptidoglycan structures. Furthermore, we discuss commonly used software that supports IM-MS data analysis of glycopeptides., (© 2024 The Authors. PROTEOMICS published by Wiley‐VCH GmbH.)

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 fully automated online enrichment and separation system for intact glycopeptides, named AutoGP, was developed in this study by integrating three different columns in a nano-LC system. Specifically, the peptide mixture from the enzymatic digestion of a complex biological sample was first loaded on a hydrophilic interaction chromatography (HILIC) column. The nonglycopeptides in the sample were washed off the column, and the glycopeptides retained by the HILIC column were eluted to a C18 trap column to achieve an automated glycopeptide enrichment. The enriched glycopeptides were further eluted to a C18 column for separation, and the separated glycopeptides were eventually analyzed by using an orbitrap mass spectrometer (MS). The optimal operating conditions for AutoGP were systemically studied, and the performance of the fully optimized AutoGP was compared with a conventional manual system used for glycopeptide analysis. The experimental evaluation shows that the total number of glycopeptides identified is at least 1.5-fold higher, and the median coefficient of variation for the analyses is at least 50% lower by using AutoGP, as compared to the results acquired by using the manual system. In addition, AutoGP can perform effective analysis even with a 1-μg sample amount, while a 10-μg sample at least will be needed by the manual system, implying an order of magnitude better sensitivity of AutoGP. All the experimental results have consistently proven that AutoGP can be used for much better characterization of intact glycopeptides.

Reproducibility is a "proteomic dream" yet to be fully realized. A typical data analysis workflow utilizing extracted ion chromatograms (XICs) often treats the information path from identification to quantification as a one-way street. Here, we propose an XIC-centric approach in which the data flow is bidirectional: identifications are used to derive XICs whose information is in turn applied to validate the identifications. In this study, we employed liquid chromatography-mass spectrometry data from glycoprotein and human hair samples to illustrate the XIC-centric concept. At the core of this approach was XIC-based monoisotope repicking. Taking advantage of the intensity information for all detected isotopes across the whole range of an XIC peak significantly improved the accuracy and uncovered misidentifications originating from monoisotope assignment mistakes. It could also rescue non-top-ranked glycopeptide hits. Identification of glycopeptides is particularly susceptible to precursor mass errors for their low abundances, large masses, and glycans differing by 1 or 2 Da easily confused as isotopes. In addition, the XIC-centric strategy significantly reduced the problem of one XIC peak associated with multiple unique identifications, a source of quantitative irreproducibility. Taken together, the proposed approach can lead to improved identification and quantification accuracy and, ultimately, enhanced reproducibility in proteomic data analyses.

Protein post-translational modifications (PTMs) are crucial in plant cellular processes, particularly in protein folding and signal transduction. N-glycosylation and phosphorylation are notably significant PTMs, playing essential roles in regulating plant responses to environmental stimuli. However, current sequential enrichment methods for simultaneous analysis of phosphoproteome and N-glycoproteome are labor-intensive and time-consuming, limiting their throughput. Addressing this challenge, this study introduces a novel tandem S-Trap-IMAC-HILIC (S-Trap: suspension trapping; IMAC: immobilized metal ion affinity chromatography; HILIC: hydrophilic interaction chromatography) strategy, termed TIMAHAC, for simultaneous analysis of plant phosphoproteomics and N-glycoproteomics. This approach integrates IMAC and HILIC into a tandem tip format, streamlining the enrichment process of phosphopeptides and N-glycopeptides. The key innovation lies in the use of a unified buffer system and an optimized enrichment sequence to enhance efficiency and reproducibility. The applicability of TIMAHAC was demonstrated by analyzing the Arabidopsis phosphoproteome and N-glycoproteome in response to abscisic acid (ABA) treatment. Up to 1954 N-glycopeptides and 11,255 phosphopeptides were identified from Arabidopsis, indicating its scalability for plant tissues. Notably, distinct perturbation patterns were observed in the phosphoproteome and N-glycoproteome, suggesting their unique contributions to ABA response. Our results reveal that TIMAHAC offers a comprehensive approach to studying complex regulatory mechanisms and PTM interplay in plant biology, paving the way for in-depth investigations into plant signaling networks., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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.)

We report the comparison of mass-spectral-based abundances of tryptic glycopeptides to fluorescence abundances of released labeled glycans and the effects of mass and charge state and in-source fragmentation on glycopeptide abundances. The primary glycoforms derived from Rituximab, NISTmAb, Evolocumab, and Infliximab were high-mannose and biantennary complex galactosylated and fucosylated N-glycans. Except for Evolocumab, in-source ions derived from the loss of HexNAc or HexNAc-Hex sugars are prominent for other therapeutic IgGs. After excluding in-source fragmentation of glycopeptide ions from the results, a linear correlation was observed between fluorescently labeled N-glycan and glycopeptide abundances over a dynamic range of 500. Different charge states of human IgG-derived glycopeptides containing a wider variety of abundant attached glycans were also investigated to examine the effects of the charge state on ion abundances. These revealed a linear dependence of glycopeptide abundance on the mass of the glycan with higher charge states favoring higher-mass glycans. Findings indicate that the mass spectrometry-based bottom-up approach can provide results as accurate as those of glycan release studies while revealing the origin of each attached glycan. These site-specific relative abundances are conveniently displayed and compared using previously described glycopeptide abundance distribution spectra "GADS" representations. Mass spectrometry data are available from the MAssIVE repository (MSV000093562).

Background: A better diagnostic marker is in need to distinguish breast cancer from suspicious breast lesions. The abnormal glycosylation of haptoglobin has been documented to assist cancer diagnosis. This study aims to evaluate disease-specific haptoglobin (DSHp)-β N-glycosylation as a potential biomarker for breast cancer diagnosis., Methods: DSHp-β chains of 497 patients with suspicious breast lesions who underwent breast surgery were separated from serum immunoinflammatory-related protein complexes. DSHp-β N-glycosylation was quantified by mass spectrometric analysis. After missing data imputation and propensity score matching, patients were randomly assigned to the training set (n = 269) and validation set (n = 113). Logistic regression analysis was employed in model and nomogram construction. The diagnostic performance was analyzed with receiver operating characteristic and calibration curves., Results: 95 N-glycopeptides at glycosylation sites N207/N211, N241, and N184 were identified in 235 patients with benign breast diseases and 262 patients with breast cancer. DSHp-β N-tetrafucosyl and hexafucosyl were significantly increased in breast cancer compared with benign diseases (p < 0.001 and p = 0.001, respectively). The new diagnostic model and nomogram included GN2F2, G6N3F6, GN2FS at N184, G-N&G2S2, G2&G3NFS, G2N3F, GN3 at N207/N211, CEA, CA153, and could reliably distinguish breast cancer from benign diseases. For the training set, validation set, and training and validation sets, the area under the curves (AUCs) were 0.80 (95% CI: 0.75-0.86, specificity: 87%, sensitivity: 62%), 0.77 (95% CI:0.69-0.86, specificity: 75%, sensitivity: 69%), and 0.80 (95% CI:0.76-0.84, specificity: 77%, sensitivity: 68%), respectively. CEA, CA153, and their combination yielded AUCs of 0.62 (95% CI: 0.56-0.67, specificity: 29%, sensitivity: 90%), 0.65 (95% CI: 0.60-0.71, specificity: 74%, sensitivity: 51%), and 0.67 (95% CI: 0.62-0.73, specificity: 60%, sensitivity: 68%), respectively., Conclusions: The combination of DSHp-β N-glycopeptides, CEA, and CA153 might be a better serologic marker to differentiate between breast cancer and benign breast diseases. The dysregulated N-glycosylation of serum DSHp-β could provide insights into breast tumorigenesis., (© 2024. The Author(s).)

Glycosylation is a key modulator of the functional state of proteins. Recent developments in large-scale analysis of intact glycopeptides have enabled the identification of numerous glycan structures that are relevant in pathophysiological processes. However, one motif found in N -glycans, poly- N -acetyllactosamine (polyLacNAc), still poses a substantial challenge to mass spectrometry-based glycoproteomic analysis due to its relatively low abundance and large size. In this work, we developed approaches for the systematic mapping of polyLacNAc-elongated N -glycans in melanoma cells. We first evaluated five anion exchange-based matrices for enriching intact glycopeptides and selected two materials that provided better overall enrichment efficiency. We then tested the robustness of the methodology by quantifying polyLacNAc-containing glycopeptides as well as changes in protein fucosylation and sialylation. Finally, we applied the optimal enrichment methods to discover glycopeptides containing polyLacNAc motifs in melanoma cells and found that integrins and tetraspanins are substantially modified with these structures. This study demonstrates the feasibility of glycoproteomic approaches for identification of glycoproteins with polyLacNAc motifs.

Protein glycosylation, a complex and heterogeneous post-translational modification that is frequently dysregulated in disease, has been difficult to analyse at scale. Here we report a data-independent acquisition technique for the large-scale mass-spectrometric quantification of glycopeptides in plasma samples. The technique, which we named 'OxoScan-MS', identifies oxonium ions as glycopeptide fragments and exploits a sliding-quadrupole dimension to generate comprehensive and untargeted oxonium ion maps of precursor masses assigned to fragment ions from non-enriched plasma samples. By applying OxoScan-MS to quantify 1,002 glycopeptide features in the plasma glycoproteomes from patients with COVID-19 and healthy controls, we found that severe COVID-19 induces differential glycosylation in IgA, haptoglobin, transferrin and other disease-relevant plasma glycoproteins. OxoScan-MS may allow for the quantitative mapping of glycoproteomes at the scale of hundreds to thousands of samples., (© 2023. The Author(s).)

LC-MS-based N -glycosylation profiling in four human serum IgG subclasses (IgG1, IgG2, IgG3, and IgG4) often requires additional affinity-based enrichment of specific IgG subclasses, owing to the high amino acid sequence similarity of Fc glycopeptides among subclasses. Notably, for IgG4 and the major allotype of IgG3, the glycopeptide precursors share identical retention time and mass and therefore cannot be distinguished based on precursor or glycan fragmentation. Here, we developed a parallel reaction monitoring (PRM)-based method for quantifying Fc glycopeptides through combined transitions generated from both glycosidic and peptide bond fragmentation. The latter enables the subpopulation of IgG3 and IgG4 to be directly distinguished according to mass differences without requiring further enrichment of specific IgG subclasses. In addition, a multinozzle electrospray emitter coupled to a capillary flow liquid chromatograph was used to increase the robustness and detection sensitivity of the method for low-yield peptide backbone fragment ions. The gradient was optimized to decrease the overall run time and make the method compatible with high-throughput analysis. We demonstrated that this method can be used to effectively monitor the relative levels of 13 representative glycoforms, with a good limit of detection for individual IgG subclasses.

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.

Glycosylation is widely recognized as the most complex post-translational modification due to the widespread presence of macro- and microheterogeneities, wherein its biological consequence is closely related to both the glycosylation sites and the glycan fine structures. Yet, efficient site-specific detailed glycan characterization remains a significant analytical challenge. Here, utilizing an Orbitrap-Omnitrap platform, higher-energy electron-activated dissociation (heExD) tandem mass spectrometry (MS/MS) revealed extraordinary efficacy for the structural characterization of intact glycopeptides. HeExD produced extensive fragmentation within both the glycan and the peptide, including A-/B-/C-/Y-/Z-/X-ions from the glycan motif and a-/b-/c-/x-/y-/z-type peptide fragments (with or without the glycan). The intensity of cross-ring cleavage and backbone fragments retaining the intact glycan was highly dependent on the electron energy. Among the four electron energy levels investigated, electronic excitation dissociation (EED) provided the most comprehensive structural information, yielding a complete series of glycosidic fragments for accurate glycan topology determination, a wealth of cross-ring fragments for linkage definition, and the most extensive peptide backbone fragments for accurate peptide sequencing and glycosylation site localization. The glycan fragments observed in the EED spectrum correlated well with the fragmentation patterns observed in EED MS/MS of the released glycans. The advantages of EED over higher-energy collisional dissociation (HCD), stepped collision energy HCD (sceHCD), and electron-transfer/higher-energy collisional dissociation (EThcD) were demonstrated for the characterization of a glycopeptide bearing a biantennary disialylated glycan. EED can produce a complete peptide backbone and glycan sequence coverage even for doubly protonated precursors. The exceptional performance of heExD MS/MS, particularly EED MS/MS, in site-specific detailed glycan characterization on an Orbitrap-Omnitrap hybrid instrument presents a novel option for in-depth glycosylation analysis.

Glycosylation affects the safety and efficacy of therapeutic proteins and is often considered a critical quality attribute (CQA). Therefore, it is important to identify and quantify glycans during drug development. Glycosylation is a highly complex post-translational modification (PTM) due to its structural heterogeneity, i.e. glycosylation site occupancy, glycan compositions, modifications, and isomers. Current analytical tools compromise either structural resolution or site specificity. Hydrophilic interaction liquid chromatography-fluorescence-mass spectrometry (HILIC-FLR-MS) is the gold standard for structural analysis of released glycans, but lacks information on site specificity and occupation. However, HILIC-FLR-MS often uses salt in the solvent, which impairs analysis robustness and sensitivity. Site-specific glycosylation analysis via glycopeptides, upon proteolytic digestion, is commonly performed by reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS), but provides only compositional and limited structural glycan information. In this study, we introduce a salt-free, glycopeptide-based HILIC-tandem mass spectrometry (HILIC-MS/MS) method that provides glycan identification, glycan isomer separation and site-specific information simultaneously. Moreover, HILIC-MS/MS demonstrated comparable relative quantification results as released glycan HILIC-FLR-MS. Further, our new method improves the retention of hydrophilic peptides, allowing simultaneous analysis of important CQAs such as deamidation in antibodies. The developed method offers a valuable tool to streamline the site-specific glycosylation analysis of glycoproteins, which is particularly important for the expanding landscape of novel therapeutic formats in the biopharmaceutical industry.