Search

Cancer early detection and treatment response prediction continue to pose significant challenges. Cancer liquid biopsies focusing on detecting circulating tumor cells (CTCs) and DNA (ctDNA) have shown enormous potential due to their non-invasive nature and the implications in precision cancer management. Recently, liquid biopsy has been further expanded to profile glycoproteins, which are the products of post-translational modifications of proteins and play key roles in both normal and pathological processes, including cancers. The advancements in chemical and mass spectrometry-based technologies and artificial intelligence-based platforms have enabled extensive studies of cancer and organ-specific changes in glycans and glycoproteins through glycomics and glycoproteomics. Glycoproteomic analysis has emerged as a promising tool for biomarker discovery and development in early detection of cancers and prediction of treatment efficacy including response to immunotherapies. These biomarkers could play a crucial role in aiding in early intervention and personalized therapy decisions. In this review, we summarize the significant advance in cancer glycoproteomic biomarker studies and the promise and challenges in integration into clinical practice to improve cancer patient care.

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The primary proteins in chicken egg white are glycoproteins, which generate substantial glycopeptides in the digestive tract following gastrointestinal digestion. The structures of the glycopeptides have yet to be identified and elucidated. In this study, chicken egg white was digested by trypsin, and N-glycopeptides were enriched from the hydrolysate by hydrophilic interaction chromatography and O-glycopeptides by retain AX chromatography combined with AminoLink resin adsorption. The structures of these glycopeptides were analyzed by liquid chromatography-mass spectrometry (LC-MS), and their potential intestinal effects were explored by bioinformatics analysis. Mass spectrometry analysis identified 1 720 N-glycopeptides from 49 N-glycoproteins and 565 O-glycopeptides from 29 O-glycoproteins. Glycopeptides derived from ovomucin were the most diverse, including 490 N-glycopeptides and 216 O-glycopeptides. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that sialylated N-glycoproteins were mainly involved in interleukin-17 signaling pathway and ferroptosis, while sialylated O-glycoproteins were mainly involved in cholesterol metabolism, vitamin digestion and absorption, the hypoxia-inducible factor-1 signaling pathway and the renin-angiotensin system. This study provides crucial structural information for further research on the potential effects of egg white glycopeptides on the intestinal tract.

Introduction: The human plasma glycoproteome holds enormous potential to identify personalized biomarkers for diagnostics. Glycoproteomics has matured into a technology for plasma N-glycoproteome analysis but further evolution towards clinical applications depends on the clinical validity and understanding of protein- and site-specific glycosylation changes in disease. Objectives: Here, we exploited the uniqueness of a patient cohort of genetic defects in well-defined glycosylation pathways to assess the clinical applicability of plasma N-glycoproteomics. Methods: Comparative glycoproteomics was performed of blood plasma from 40 controls and 74 patients with 13 different genetic diseases that impact the protein N-glycosylation pathway. Baseline glycosylation in healthy individuals was compared to reference glycome and intact transferrin protein mass spectrometry data. Use of glycoproteomics data for biomarker discovery and sample stratification was evaluated by multivariate chemometrics and supervised machine learning. Clinical relevance of site-specific glycosylation changes were evaluated in the context of genetic defects that lead to distinct accumulation or loss of specific glycans. Integrated analysis of site-specific glycoproteome changes in disease was performed using chord diagrams and correlated with intact transferrin protein mass spectrometry data. Results: Glycoproteomics identified 191 unique glycoforms from 58 unique peptide sequences of 34 plasma glycoproteins that span over 3 magnitudes of abundance in plasma. Chemometrics identified high-specificity biomarker signatures for each of the individual genetic defects with better stratification performance than the current diagnostic standard method. Bioinformatic analyses revealed site-specific glycosylation differences that could be explained by underlying glycobiology and protein-intrinsic factors. Conclusion: Our work illustrates the strong potential of plasma glycoproteomics to significantly increase specificity of glycoprotein biomarkers with direct insights in site-specific glycosylation changes to better understand the glycobiological mechanisms underlying human disease.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Protein cysteine S-glycosylation is a relatively rare and less well characterized post-translational modification (PTM). Creating reliable model proteins that carry this modification is challenging. The lack of available models or natural S-glycosylated proteins significantly hampers the development of mass-spectrometry-based (MS-based) methodologies for detecting protein cysteine S-glycosylation in real-world proteomic studies. There is also limited MS-sequencing data describing it as easier to create synthetic S-glycopeptides. Here, we present the results of an in-depth manual analysis of automatically annotated CID/HCD spectra for model S-glucopeptides. The CID spectra show a long series of y/b-fragment ions with retained S-glucosylation, regardless of the dominant m/z signals corresponding to neutral loss of 1,2-anhydroglucose from the precursor ions. In addition, the spectra show signals manifesting glucosyl transfer from the cysteine position onto lysine, arginine (Lys, Arg) side chains, and a peptide N-terminus. Other spectral evidence indicates that the N-glucosylated initial products of transfer are converted into N-fructosylated (i.e., glycated) structures due to Amadori rearrangement. We discuss the peculiar transfer of the glucose oxocarbenium ion (Glc+) to positively charged guanidinium residue (ArgH+) and propose a mechanism for the gas-phase Amadori rearrangement involving a 1,2-hydride ion shift. [ABSTRACT FROM AUTHOR]

[Display omitted] • Plasma glycoproteomics provides broad and reproducible coverage for clinical applications. • Chemometric analyses demonstrates great potential of glycoproteomics for biomarker applications. • Site-specific glycosylation changes delineate glycobiology processes underlying disease. • Bioinformatic analyses of disease samples enable integral interpretation of glycoproteomes. • Glycosylation changes can be site-specific on single protein molecules depending on disease. The human plasma glycoproteome holds enormous potential to identify personalized biomarkers for diagnostics. Glycoproteomics has matured into a technology for plasma N-glycoproteome analysis but further evolution towards clinical applications depends on the clinical validity and understanding of protein- and site-specific glycosylation changes in disease. Here, we exploited the uniqueness of a patient cohort of genetic defects in well-defined glycosylation pathways to assess the clinical applicability of plasma N-glycoproteomics. Comparative glycoproteomics was performed of blood plasma from 40 controls and 74 patients with 13 different genetic diseases that impact the protein N-glycosylation pathway. Baseline glycosylation in healthy individuals was compared to reference glycome and intact transferrin protein mass spectrometry data. Use of glycoproteomics data for biomarker discovery and sample stratification was evaluated by multivariate chemometrics and supervised machine learning. Clinical relevance of site-specific glycosylation changes were evaluated in the context of genetic defects that lead to distinct accumulation or loss of specific glycans. Integrated analysis of site-specific glycoproteome changes in disease was performed using chord diagrams and correlated with intact transferrin protein mass spectrometry data. Glycoproteomics identified 191 unique glycoforms from 58 unique peptide sequences of 34 plasma glycoproteins that span over 3 magnitudes of abundance in plasma. Chemometrics identified high-specificity biomarker signatures for each of the individual genetic defects with better stratification performance than the current diagnostic standard method. Bioinformatic analyses revealed site-specific glycosylation differences that could be explained by underlying glycobiology and protein-intrinsic factors. Our work illustrates the strong potential of plasma glycoproteomics to significantly increase specificity of glycoprotein biomarkers with direct insights in site-specific glycosylation changes to better understand the glycobiological mechanisms underlying human disease. [ABSTRACT FROM AUTHOR]

Traditional methodologies often fall short in addressing the complexity of biological systems. In this regard, system biology omics have brought invaluable tools for conducting comprehensive analysis. Current sequencing capabilities have revolutionized genetics and genomics studies, as well as the characterization of transcriptional profiling and dynamics of several species and sample types. Biological systems experience complex biochemical processes involving thousands of molecules. These processes occur at different levels that can be studied using mass spectrometry-based (MS-based) analysis, enabling high-throughput proteomics, glycoproteomics, glycomics, metabolomics, and lipidomics analysis. Here, we present the most up-to-date techniques utilized in the completion of omics analysis. Additionally, we include some interesting examples of the applicability of multi omics to a variety of biological systems.

Abstract Background Glycosylation is an enzyme-catalyzed post-translational modification that is distinct from glycation and is present on a majority of plasma proteins. N-glycosylation occurs on asparagine residues predominantly within canonical N-glycosylation motifs (Asn-X-Ser/Thr) although non-canonical N-glycosylation motifs Asn-X-Cys/Val have also been reported. Albumin is the most abundant protein in plasma whose glycation is well-studied in diabetes mellitus. However, albumin has long been considered a non-glycosylated protein due to absence of canonical motifs. Albumin contains two non-canonical N-glycosylation motifs, of which one was recently reported to be glycosylated. Methods We enriched abundant serum proteins to investigate their N-linked glycosylation followed by trypsin digestion and glycopeptide enrichment by size-exclusion or mixed-mode anion-exchange chromatography. Glycosylation at canonical as well as non-canonical sites was evaluated by liquid chromatography–tandem mass spectrometry (LC–MS/MS) of enriched glycopeptides. Deglycosylation analysis was performed to confirm N-linked glycosylation at non-canonical sites. Albumin-derived glycopeptides were fragmented by MS3 to confirm attached glycans. Parallel reaction monitoring was carried out on twenty additional samples to validate these findings. Bovine and rabbit albumin-derived glycopeptides were similarly analyzed by LC–MS/MS. Results Human albumin is N-glycosylated at two non-canonical sites, Asn68 and Asn123. N-glycopeptides were detected at both sites bearing four complex sialylated glycans and validated by MS3-based fragmentation and deglycosylation studies. Targeted mass spectrometry confirmed glycosylation in twenty additional donor samples. Finally, the highly conserved Asn123 in bovine and rabbit serum albumin was also found to be glycosylated. Conclusions Albumin is a glycoprotein with conserved N-linked glycosylation sites that could have potential clinical applications.

Abstract Background Hepatocellular carcinoma (HCC) ranks as the third most common cause of cancer related death globally, representing a substantial challenge to global healthcare systems. In China, the primary risk factor for HCC is the hepatitis B virus (HBV). Aberrant serum glycoconjugate levels have long been linked to the progression of HBV-associated HCC (HBV-HCC). Nevertheless, few study systematically explored the dysregulation of glycoconjugates in the progression of HBV-associated HCC and their potency as the diagnostic and prognostic biomarker. Methods An integrated strategy that combined transcriptomics, glycomics, and glycoproteomics was employed to comprehensively investigate the dynamic alterations in glyco-genes, N-glycans, and glycoproteins in the progression of HBV- HCC. Results Bioinformatic analysis of Gene Expression Omnibus (GEO) datasets uncovered dysregulation of fucosyltransferases (FUTs) in liver tissues from HCC patients compared to adjacent tissues. Glycomic analysis indicated an elevated level of fucosylated N-glycans, especially a progressive increase in fucosylation levels on IgA1 and IgG2 determined by glycoproteomic analysis. Conclusions The findings indicate that the abnormal fucosylation plays a pivotal role in the progression of HBV-HCC. Systematic and integrative multi-omic analysis is anticipated to facilitate the discovery of aberrant glycoconjugates in tumor progression.

Lung cancer is the leading cause of cancer death and non-small cell lung carcinoma (NSCLC) accounting for majority of lung cancers. Thus, it is important to find potential biomarkers, such as glycans and glycoproteins, which can be used as diagnostic tools against NSCLC. Here, the N-glycome, proteome, and N-glycosylation distribution maps of tumor and peritumoral tissues of Filipino lung cancer patients (n = 5) were characterized. We present several case studies with varying stages of cancer development (I-III), mutation status (EGFR, ALK), and biomarker expression based on a three-gene panel (CD133, KRT19, and MUC1). Although the profiles of each patient were unique, specific trends arose that correlated with the role of aberrant glycosylation in cancer progression. Specifically, we observed a general increase in the relative abundance of high-mannose and sialofucosylated N-glycans in tumor samples. Analysis of the glycan distribution per glycosite revealed that these sialofucosylated N-glycans were specifically attached to glycoproteins involved in key cellular processes, including metabolism, cell adhesion, and regulatory pathways. Protein expression profiles showed significant enrichment of dysregulated proteins involved in metabolism, adhesion, cell-ECM interactions, and N-linked glycosylation, supporting the protein glycosylation results. The present case series study provides the first demonstration of a multi-platform mass-spectrometric analysis specifically for Filipino lung cancer patients.

Glycans are complex biomolecules that encode rich information and regulate various biological processes, such as fertilization, host‐pathogen binding, and immune recognition, through interactions with glycan‐binding proteins. A key driving force for glycan‐protein recognition is the interaction between the π electron density of aromatic amino acid side chains and polarized C─H groups of the pyranose (termed the CH–π interaction). However, the relatively weak binding affinity between glycans and proteins has hindered the application of glycan detection and imaging. Here, computational modeling and molecular dynamics simulations are employed to design a chemical strategy that enhances the CH–π interaction between glycans and proteins by genetically incorporating electron‐rich tryptophan derivatives into a lectin PhoSL, which specifically recognizes core fucosylated N‐linked glycans. This significantly enhances the binding affinity of PhoSL with the core fucose ligand and enables sensitive detection and imaging of core fucosylated glycans in vitro and in xenograft tumors in mice. Further, the study showed that this strategy is applicable to improve the binding affinity of GafD lectin for N‐acetylglucosamine‐containing glycans. The approach thus provides a general and effective way to manipulate glycan‐protein recognition for glycoscience applications. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Human noroviruses, globally the main cause of viral gastroenteritis, show strain specific affinity for histo-blood group antigens (HBGA) and can successfully be propagated ex vivo in human intestinal enteroids (HIEs). HIEs established from jejunal stem cells of individuals with different ABO, Lewis and secretor geno- and phenotypes, show varying susceptibility to such infections. Using bottom-up glycoproteomic approaches we have defined and compared the N -linked glycans of glycoproteins of seven jejunal HIEs. Membrane proteins were extracted, trypsin digested, and glycopeptides enriched by hydrophilic interaction liquid chromatography and analyzed by nanoLC-MS/MS. The Byonic software was used for glycopeptide identification followed by hands-on verifications and interpretations. Glycan structures and attachment sites were identified from MS2 spectra obtained by higher-energy collision dissociation through analysis of diagnostic saccharide oxonium ions (B-ions), stepwise glycosidic fragmentation of the glycans (Y-ions), and peptide sequence ions (b- and y-ions). Altogether 694 unique glycopeptides from 93 glycoproteins were identified. The N-glycans encompassed pauci- and oligomannose, hybrid- and complex-type structures. Notably, polyfucosylated HBGA-containing glycopeptides of the four glycoproteins tetraspanin-8, carcinoembryonic antigen-related cell adhesion molecule 5, sucrose-isomaltase and aminopeptidase N were especially prominent and were characterized in detail and related to donor ABO, Lewis and secretor types of each HIE. Virtually no sialylated N-glycans were identified for these glycoproteins suggesting that terminal sialylation was infrequent compared to fucosylation and HBGA biosynthesis. This approach gives unique site-specific information on the structural complexity of N-linked glycans of glycoproteins of human HIEs and provides a platform for future studies on the role of host glycoproteins in gastrointestinal infectious diseases. [ABSTRACT FROM AUTHOR]

Circulating tumor cells (CTCs) are cancer cells that detach from the original site and reach the bloodstream. The most aggressive CTCs survive various immune system attacks and initiate metastasis formation. Importantly, CTCs are not specifically targeted by the current immunotherapies due to the limited knowledge on specific targets. Proteomic profiling can be a powerful tool for understanding some of the immune evasion mechanisms used by cancer cells and particularly CTCs. These mechanisms are generally linked to the expression of specific surface proteins/peptides (i.e. the surfaceome). The study of the peptides that bind to class I molecules of the major histocompatibility complex (MHC‐I) and of the various glycoproteins expressed on CTC surface may open a completely new avenue for the discovery of novel mechanisms of immune evasion. In this review, we discuss how immunopeptidomic and glycoproteomic studies of CTCs that interact with immune cells could help to better understand how metastasis‐initiator CTCs escape the host immune response. We also describe how immunopeptidomic and glycoproteomic studies are carried out. [ABSTRACT FROM AUTHOR]

Background: Glycosylation is an enzyme-catalyzed post-translational modification that is distinct from glycation and is present on a majority of plasma proteins. N-glycosylation occurs on asparagine residues predominantly within canonical N-glycosylation motifs (Asn-X-Ser/Thr) although non-canonical N-glycosylation motifs Asn-X-Cys/Val have also been reported. Albumin is the most abundant protein in plasma whose glycation is well-studied in diabetes mellitus. However, albumin has long been considered a non-glycosylated protein due to absence of canonical motifs. Albumin contains two non-canonical N-glycosylation motifs, of which one was recently reported to be glycosylated. Methods: We enriched abundant serum proteins to investigate their N-linked glycosylation followed by trypsin digestion and glycopeptide enrichment by size-exclusion or mixed-mode anion-exchange chromatography. Glycosylation at canonical as well as non-canonical sites was evaluated by liquid chromatography–tandem mass spectrometry (LC–MS/MS) of enriched glycopeptides. Deglycosylation analysis was performed to confirm N-linked glycosylation at non-canonical sites. Albumin-derived glycopeptides were fragmented by MS3 to confirm attached glycans. Parallel reaction monitoring was carried out on twenty additional samples to validate these findings. Bovine and rabbit albumin-derived glycopeptides were similarly analyzed by LC–MS/MS. Results: Human albumin is N-glycosylated at two non-canonical sites, Asn68 and Asn123. N-glycopeptides were detected at both sites bearing four complex sialylated glycans and validated by MS3-based fragmentation and deglycosylation studies. Targeted mass spectrometry confirmed glycosylation in twenty additional donor samples. Finally, the highly conserved Asn123 in bovine and rabbit serum albumin was also found to be glycosylated. Conclusions: Albumin is a glycoprotein with conserved N-linked glycosylation sites that could have potential clinical applications. [ABSTRACT FROM AUTHOR]

Background: Hepatocellular carcinoma (HCC) ranks as the third most common cause of cancer related death globally, representing a substantial challenge to global healthcare systems. In China, the primary risk factor for HCC is the hepatitis B virus (HBV). Aberrant serum glycoconjugate levels have long been linked to the progression of HBV-associated HCC (HBV-HCC). Nevertheless, few study systematically explored the dysregulation of glycoconjugates in the progression of HBV-associated HCC and their potency as the diagnostic and prognostic biomarker. Methods: An integrated strategy that combined transcriptomics, glycomics, and glycoproteomics was employed to comprehensively investigate the dynamic alterations in glyco-genes, N-glycans, and glycoproteins in the progression of HBV- HCC. Results: Bioinformatic analysis of Gene Expression Omnibus (GEO) datasets uncovered dysregulation of fucosyltransferases (FUTs) in liver tissues from HCC patients compared to adjacent tissues. Glycomic analysis indicated an elevated level of fucosylated N-glycans, especially a progressive increase in fucosylation levels on IgA1 and IgG2 determined by glycoproteomic analysis. Conclusions: The findings indicate that the abnormal fucosylation plays a pivotal role in the progression of HBV-HCC. Systematic and integrative multi-omic analysis is anticipated to facilitate the discovery of aberrant glycoconjugates in tumor progression. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) screening relies primarily on stool analysis to identify occult blood. However, its sensitivity for detecting precancerous lesions is limited, requiring the development of new tools to improve CRC screening. Carcinogenesis involves significant alterations in mucosal epithelium glycocalyx that decisively contribute to disease progression. Building on this knowledge, we examined patient series comprehending premalignant lesions, colorectal tumors, and healthy controls for the T-antigen—a short-chain O-glycosylation of proteins considered a surrogate marker of malignancy in multiple solid cancers. We found the T-antigen in the secretions of dysplastic lesions as well as in cancer. In CRC, T-antigen expression was associated with the presence of distant metastases. In parallel, we analyzed a broad number of stools from individuals who underwent colonoscopy, which showed high T expressions in high-grade dysplasia and carcinomas. Employing mass spectrometry-based lectin-affinity enrichment, we identified a total of 262 proteins, 67% of which potentially exhibited altered glycosylation patterns associated with cancer and advanced pre-cancerous lesions. Also, we found that the stool (glyco)proteome of pre-cancerous lesions is enriched for protein species involved in key biological processes linked to humoral and innate immune responses. This study offers a thorough analysis of the stool glycoproteome, laying the groundwork for harnessing glycosylation alterations to improve non-invasive cancer detection. [ABSTRACT FROM AUTHOR]

Modern glycoproteomics experiments require the use of search engines due to the generation of countless spectra. While these tools are valuable, manual validation of search engine results is often required for detailed analysis of glycopeptides as false-discovery rates are often not reliable for glycopeptide data. Near-isobaric mismatches are a common source of misidentifications for the popular glycopeptide-focused search engine pGlyco3.0, and in this technical note we share a strategy and script that improves the accuracy of the search utilizing two manually validated datasets of the glycoproteins CD16a and HIV-1 Env as proof-of-principle. [ABSTRACT FROM AUTHOR]

Abstract Cancer early detection and treatment response prediction continue to pose significant challenges. Cancer liquid biopsies focusing on detecting circulating tumor cells (CTCs) and DNA (ctDNA) have shown enormous potential due to their non-invasive nature and the implications in precision cancer management. Recently, liquid biopsy has been further expanded to profile glycoproteins, which are the products of post-translational modifications of proteins and play key roles in both normal and pathological processes, including cancers. The advancements in chemical and mass spectrometry-based technologies and artificial intelligence-based platforms have enabled extensive studies of cancer and organ-specific changes in glycans and glycoproteins through glycomics and glycoproteomics. Glycoproteomic analysis has emerged as a promising tool for biomarker discovery and development in early detection of cancers and prediction of treatment efficacy including response to immunotherapies. These biomarkers could play a crucial role in aiding in early intervention and personalized therapy decisions. In this review, we summarize the significant advance in cancer glycoproteomic biomarker studies and the promise and challenges in integration into clinical practice to improve cancer patient care.

The sample preparation step is pivotal in glycoproteomic analysis. An effective approach in glycoprotein sample preparation involves enriching glycopeptides by solid-phase extraction (SPE) using polar stationary phases in hydrophilic interaction liquid chromatography (HILIC) mode. The aim of this work is to show how different experimental conditions influence the enrichment efficiency of glycopeptides from human immunoglobulin G (IgG) on an aminopropyl-modified SPE column. Different compositions of the elution solvent (acetonitrile, methanol, and isopropanol), along with varying concentrations of elution solvent acidifiers (formic and acetic acid), and different concentrations of acetonitrile for the conditioning and washing solvents (65%, 75%, and 85% acetonitrile) were tested to observe their effects on the glycopeptide enrichment process. Isopropanol proved less effective in enriching glycopeptides, while acetonitrile was the most efficient, with methanol in between. Higher formic acid concentrations in the elution solvent weakened the ionic interactions, particularly with sialylated glycopeptides. Substituting formic acid with acetic acid led to earlier elution of more glycopeptides. The acetonitrile concentration in conditioning and washing solutions played a key role; at 65% acetonitrile, glycopeptides were not retained on the SPE column and were detected in the flow-through fraction. Ultimately, it was proven that the enrichment method was applicable to human plasma samples, resulting in a significant decrease in the abundances of non-glycosylated peptides. To the best of our knowledge, this study represents the first systematic investigation into the impact of the mobile phase on glycopeptide enrichment using an aminopropyl-modified SPE column in HILIC mode. This study demonstrates the substantial impact of even minor variations in experimental conditions, which have not yet been considered in the literature, on SPE-HILIC glycopeptide enrichment. Consequently, meticulous optimization of these conditions is imperative to enhance the specificity and selectivity of glycoproteomic analysis, ensuring accurate and reliable quantification. [ABSTRACT FROM AUTHOR]

Muscle-invasive bladder cancer (MIBC) remains a pressing health concern due to conventional treatment failure and significant molecular heterogeneity, hampering the development of novel targeted therapeutics. In our quest for novel targetable markers, recent glycoproteomics and bioinformatics data have pinpointed (glucose transporter 1) GLUT1 as a potential biomarker due to its increased expression in tumours compared to healthy tissues. This study explores this hypothesis in more detail, with emphasis on GLUT1 glycosylation patterns and cancer specificity. Immunohistochemistry analysis across a diverse set of human bladder tumours representing all disease stages revealed increasing GLUT1 expression with lesion severity, extending to metastasis, while remaining undetectable in healthy urothelium. In line with this, GLUT1 emerged as a marker of reduced overall survival. Revisiting nanoLC-EThcD-MS/MS data targeting immature O-glycosylation on muscle-invasive tumours identified GLUT1 as a carrier of short glycosylation associated with invasive disease. Precise glycosite mapping uncovered significant heterogeneity between patient samples, but also common glycopatterns that could provide the molecular basis for targeted solutions. Immature O-glycosylation conferred cancer specificity to GLUT1, laying the molecular groundwork for enhanced targeted therapeutics in bladder cancer. Future studies should focus on a comprehensive mapping of GLUT1 glycosites for highly specific cancer-targeted therapy development for bladder cancer. [ABSTRACT FROM AUTHOR]

Glycoproteomics has accelerated in recent decades owing to numerous innovations in the analytical workflow. In particular, new mass spectrometry strategies have contributed to inroads in O-glycoproteomics, a field that lags behind N-glycoproteomics due to several unique challenges associated with the complexity of O-glycosylation. This review will focus on progress in sample preparation, enrichment strategies, and MS/MS techniques for the identification and characterization of O-glycoproteins. [ABSTRACT FROM AUTHOR]

Influenza A virus (IAV) pandemics result from interspecies transmission events within the avian reservoir and further into mammals including humans. Receptor incompatibility due to differently expressed glycan structures between species has been suggested to limit zoonotic IAV transmission from the wild bird reservoir as well as between different bird species. Using glycoproteomics, we have studied the repertoires of expressed glycan structures with focus on putative sialic acid-containing glycan receptors for IAV in mallard, chicken and tufted duck; three bird species with different roles in the zoonotic ecology of IAV. The methodology used pinpoints specific glycan structures to specific glycosylation sites of identified glycoproteins and was also used to successfully discriminate α2-3- from α2-6-linked terminal sialic acids by careful analysis of oxonium ions released from glycopeptides in tandem MS/MS (MS2), and MS/MS/MS (MS3). Our analysis clearly demonstrated that all three bird species can produce complex N-glycans including α2-3-linked sialyl Lewis structures, as well as both N- and O- glycans terminated with both α2-3- and α2-6-linked Neu5Ac. We also found the recently identified putative IAV receptor structures, Man-6P N-glycopeptides, in all tissues of the three bird species. Furthermore, we found many similarities in the repertoires of expressed receptors both between the bird species investigated and to previously published data from pigs and humans. Our findings of sialylated glycan structures, previously anticipated to be mammalian specific, in all three bird species may have major implications for our understanding of the role of receptor incompatibility in interspecies transmission of IAV. [ABSTRACT FROM AUTHOR]

Objective and design: Pancreatic cancer is a highly malignant tumor that is well known for its poor prognosis. Based on glycosylation, we performed integrated quantitative N-glycoproteomics to investigate the synergistic anti-tumor effects of aspirin and gemcitabine on pancreatic cancer cells and explore the potential molecular mechanisms of chemotherapy in pancreatic cancer. Methods and results: Two pancreatic cancer cell lines (PANC-1 and BxPC-3) were treated with gemcitabine, aspirin, and a combination (gemcitabine + aspirin). We found that the addition of aspirin enhanced the inhibitory effect of gemcitabine on the activity of PANC-1 and BxPC-3 cells. Quantitative N-glycoproteome, proteome, phosphorylation, and transcriptome data were obtained from integrated multi-omics analysis to evaluate the anti-tumor effects of aspirin and gemcitabine on pancreatic cancer cells. Mfuzz analysis of intact N-glycopeptide profiles revealed two consistent trends associated with the addition of aspirin, which showed a strong relationship between N-glycosylation and the synergistic effect of aspirin. Further analysis demonstrated that the dynamic regulation of sialylation and high-mannose glycoforms on ECM-related proteins (LAMP1, LAMP2, ITGA3, etc.) was a significant factor for the ability of aspirin to promote the anti-tumor activity of gemcitabine and the drug resistance of pancreatic cancer cells. Conclusions: In-depth analysis of N-glycosylation-related processes and pathways in pancreatic cancer cells can provide new insight for future studies regarding pancreatic cancer therapeutic targets and drug resistance mechanisms. [ABSTRACT FROM AUTHOR]

The emergence of SARS-CoV-2 variants alters the efficacy of existing immunity towards the viral spike protein, whether acquired from infection or vaccination. Mutations that impact N -glycosylation of spike may be particularly important in influencing antigenicity, but their consequences are difficult to predict. Here, we compare the glycosylation profiles and antigenicity of recombinant viral spike of ancestral Wu-1 and the Gamma strain, which has two additional N -glycosylation sites due to amino acid substitutions in the N-terminal domain (NTD). We found that a mutation at residue 20 from threonine to asparagine within the NTD caused the loss of NTD-specific antibody COVA2-17 binding. Glycan site-occupancy analyses revealed that the mutation resulted in N -glycosylation switching to the new sequon at N20 from the native N17 site. Site-specific glycosylation profiles demonstrated distinct glycoform differences between Wu-1, Gamma, and selected NTD variant spike proteins, but these did not affect antibody binding. Finally, we evaluated the specificity of spike proteins against convalescent COVID-19 sera and found reduced cross-reactivity against some mutants, but not Gamma spike compared to Wuhan spike. Our results illustrate the impact of viral divergence on spike glycosylation and SARS-CoV-2 antibody binding profiles. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Zhejiang University: Science B is the property of Springer Nature and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Abstract Glycans are complex biomolecules that encode rich information and regulate various biological processes, such as fertilization, host‐pathogen binding, and immune recognition, through interactions with glycan‐binding proteins. A key driving force for glycan‐protein recognition is the interaction between the π electron density of aromatic amino acid side chains and polarized C─H groups of the pyranose (termed the CH–π interaction). However, the relatively weak binding affinity between glycans and proteins has hindered the application of glycan detection and imaging. Here, computational modeling and molecular dynamics simulations are employed to design a chemical strategy that enhances the CH–π interaction between glycans and proteins by genetically incorporating electron‐rich tryptophan derivatives into a lectin PhoSL, which specifically recognizes core fucosylated N‐linked glycans. This significantly enhances the binding affinity of PhoSL with the core fucose ligand and enables sensitive detection and imaging of core fucosylated glycans in vitro and in xenograft tumors in mice. Further, the study showed that this strategy is applicable to improve the binding affinity of GafD lectin for N‐acetylglucosamine‐containing glycans. The approach thus provides a general and effective way to manipulate glycan‐protein recognition for glycoscience applications.

Cancer progression is linked to aberrant protein glycosylation due to the overexpression of several glycosylation enzymes. These enzymes are underexploited as potential anticancer drug targets and the development of rapid-screening methods and identification of glycosylation inhibitors are highly sought. An integrated bioinformatics and mass spectrometry-based glycomics-driven glycoproteomics analysis pipeline was performed to identify an N-glycan inhibitor against lung cancer cells. Combined network pharmacology and in silico screening approaches were used to identify a potential inhibitor, pictilisib, against several glycosylation-related proteins, such as Alpha1-6FucT, GlcNAcT-V, and Alpha2,6-ST-I. A glycomics assay of lung cancer cells treated with pictilisib showed a significant reduction in the fucosylation and sialylation of N-glycans, with an increase in high mannose-type glycans. Proteomics analysis and in vitro assays also showed significant upregulation of the proteins involved in apoptosis and cell adhesion, and the downregulation of proteins involved in cell cycle regulation, mRNA processing, and protein translation. Site-specific glycoproteomics analysis further showed that glycoproteins with reduced fucosylation and sialylation were involved in apoptosis, cell adhesion, DNA damage repair, and chemical response processes. To determine how the alterations in N-glycosylation impact glycoprotein dynamics, modeling of changes in glycan interactions of the ITGA5-ITGB1 (Integrin alpha 5-Integrin beta-1) complex revealed specific glycosites at the interface of these proteins that, when highly fucosylated and sialylated, such as in untreated A549 cells, form greater hydrogen bonding interactions compared to the high mannose-types in pictilisib-treated A549 cells. This study highlights the use of mass spectrometry to identify a potential glycosylation inhibitor and assessment of its impact on cell surface glycoprotein abundance and protein-protein interaction.

Glycosylation is the most common form of post-translational modification of proteins, critically affecting their structure and function. Using liquid chromatography and mass spectrometry for high-resolution site-specific quantification of glycopeptides coupled with high-throughput artificial intelligence-powered data processing, we analyzed differential protein glycoisoform distributions of 597 abundant serum glycopeptides and nonglycosylated peptides in 50 individuals who had been seriously ill with COVID-19 and in 22 individuals who had recovered after an asymptomatic course of COVID-19. As additional comparison reference phenotypes, we included 12 individuals with a history of infection with a common cold coronavirus, 16 patients with bacterial sepsis, and 15 healthy subjects without history of coronavirus exposure. We found statistically significant differences, at FDR < 0.05, for normalized abundances of 374 of the 597 peptides and glycopeptides interrogated between symptomatic and asymptomatic COVID-19 patients. Similar statistically significant differences were seen when comparing symptomatic COVID-19 patients to healthy controls (350 differentially abundant peptides and glycopeptides) and common cold coronavirus seropositive subjects (353 differentially abundant peptides and glycopeptides). Among healthy controls and sepsis patients, 326 peptides and glycopeptides were found to be differentially abundant, of which 277 overlapped with biomarkers that showed differential expression between symptomatic COVID-19 cases and healthy controls. Among symptomatic COVID-19 cases and sepsis patients, 101 glycopeptide and peptide biomarkers were found to be statistically significantly abundant. Using both supervised and unsupervised machine learning techniques, we found specific glycoprotein profiles to be strongly predictive of symptomatic COVID-19 infection. LASSO-regularized multivariable logistic regression and K-means clustering yielded accuracies of 100% in an independent test set and of 96% overall, respectively. Our findings are consistent with the interpretation that a majority of glycoprotein modifications observed which are shared among symptomatic COVID-19 and sepsis patients likely represent a generic consequence of a severe systemic immune and inflammatory state. However, there are glycoisoform changes that are specific and particular to severe COVID-19 infection. These may be representative of either COVID-19-specific consequences or susceptibility to or predisposition for a severe course of the disease. Our findings support the potential value of glycoproteomic biomarkers in the biomedical understanding and, potentially, the clinical management of serious acute infectious conditions.

BackgroundGlycoproteomics deals with glycoproteins that are formed by post-translational modification when sugars (like fucose and sialic acid) are attached to protein. Glycosylation of proteins influences function, but whether glycosylation is altered by diet is unknown.ObjectiveTo evaluate the effect of consuming a diet based on the Dietary Guidelines for Americans on circulating glycoproteins that have previously been associated with cardiometabolic diseases.DesignForty-four women, with one or more metabolic syndrome characteristics, completed an 8-week randomized controlled feeding intervention (n = 22) consuming a diet based on the Dietary Guidelines for Americans (DGA 2010); the remaining consumed a 'typical American diet' (TAD, n = 22). Fasting serum samples were obtained at week0 (baseline) and week8 (post-intervention); 17 serum proteins were chosen for targeted analyses. Protein standards and serum samples were analyzed in a UHPLC-MS protocol to determine peptide concentration and their glycan (fucosylation or sialylation) profiles. Data at baseline were used in correlational analyses; change in proteins and glycans following intervention were used in non-parametric analyses.ResultsAt baseline, women with more metabolic syndrome characteristics had more fucosylation (total di-fucosylated proteins: p = 0.045) compared to women with a lesser number of metabolic syndrome characteristics. Dietary refined grain intake was associated with increased total fucosylation (ρ = - 0.530, p

Immunopeptidomics, the study of peptide antigens presented on the cell surface by the major histocompatibility complex (MHC), offers insights into how our immune system recognises self/non-self in health and disease. We recently discovered that hyper-processed (remodelled) N-glycans are dominant features decorating viral spike immunopeptides presented via MHC-class II (MHC-II) molecules by dendritic cells pulsed with SARS-CoV-2 spike protein, but it remains unknown if endogenous immunopeptides also undergo N-glycan remodelling. Taking a multi-omics approach, we here interrogate published MHC-II immunopeptidomics datasets of cultured monocyte-like (THP-1) and breast cancer-derived (MDA-MB-231) cell lines for overlooked N-glycosylated peptide antigens, which we compare to their source proteins in the cellular glycoproteome using proteomics and N-glycomics data from matching cell lines. Hyper-processed chitobiose core and paucimannosidic N-glycans alongside under-processed oligomannosidic N-glycans were found to prevalently modify MHC-II-bound immunopeptides isolated from both THP-1 and MDA-MB-231, while complex/hybrid-type N-glycans were (near-)absent in the immunopeptidome as supported further by new N-glycomics data generated from isolated MHC-II-bound peptides derived from MDA-MB-231 cells. Contrastingly, the cellular proteomics and N-glycomics data from both cell lines revealed conventional N-glycosylation rich in complex/hybrid-type N-glycans, which, together with the identification of key lysosomal glycosidases, suggest that MHC-II peptide antigen processing is accompanied by extensive N-glycan trimming. N-glycan remodelling appeared particularly dramatic for cell surface-located glycoproteins while less remodelling was observed for lysosomal-resident glycoproteins. Collectively, our findings indicate that both under- and hyper-processed N-glycans are prevalent features of endogenous MHC-II immunopeptides, an observation that demands further investigation to enable a better molecular-level understanding of immune surveillance. [ABSTRACT FROM AUTHOR]

Proteoglycans are a small but diverse family of proteins that play a wide variety of roles at the cell surface and in the extracellular matrix. In addition to their glycosaminoglycan (GAG) chains, they are N- and O-glycosylated. All of these types of glycosylation are crucial to their function but present a considerable analytical challenge. We describe the combination of serial proteolysis followed by the application of higher-energy collisional dissociation (HCD) and electron transfer/higher-energy collisional dissociation (EThcD) to optimize protein sequence coverage and glycopeptide identification from proteoglycans. In many cases, the use of HCD alone allows the identification of more glycopeptides. However, the localization of glycoforms on multiply glycosylated peptides has remained elusive. We demonstrate the use of EThcD for the confident assignment of glycan compositions on multiply glycosylated peptides. Dense glycosylation on proteoglycans is key to their biological function; thus, developing tools to identify and quantify doubly glycosylated peptides is of interest. Additionally, glycoproteomics searches identify glycopeptides in otherwise poorly covered regions of proteoglycans. The development of these and other analytical tools may permit glycoproteomic similarity comparisons in biological samples. [ABSTRACT FROM AUTHOR]

Background: There is a need for new serum biomarkers for early detection of hepatocellular carcinoma (HCC). Haptoglobin (Hp) N-glycosylation is altered in HCC, but the diagnostic value of site-specific Hp glycobiomarkers is rarely reported. We aimed to determine the site-specific glycosylation profile of Hp for early-stage HCC diagnosis. Method: Hp glycosylation was analyzed in the plasma of patients with liver diseases (n=57; controls), early-stage HCC (n=50) and late-stage HCC (n=32). Hp phenotype was determined by immunoblotting. Hp was immunoisolated and digested into peptides. N-glycopeptides were identified and quantified using liquid chromatography-mass spectrometry. Cohort samples were compared using Wilcoxon rank-sum (Mann-Whitney U) tests. Diagnostic performance was assessed using receiver operating characteristic (ROC) curves and area under curve (AUC). Results: Significantly higher fucosylation, branching and sialylation of Hp glycans, and expression of high-mannose glycans, was observed as disease progressed from cirrhosis to early- and late-stage HCC. Several glycopeptides demonstrated high values for early diagnosis of HCC, with an AUC of 93% (n=1), >80% (n=3), >75% (n=13) and >70% (n=11), compared with alpha-fetoprotein (AFP; AUC of 79%). The diagnostic performance of the identified biomarkers was only slightly affected by Hp phenotype. Conclusion: We identified a panel of Hp glycopeptides that are significantly differentially regulated in early- and late-stage HCC. Some glycobiomarkers exceeded the diagnostic value of AFP (the most commonly used biomarker for HCC diagnosis). Our findings provide evidence that glycobiomarkers can be effective in the diagnosis of early HCC - individually, as a panel of glycopeptides or combined with conventional serological biomarkers. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) signifies a significant healthcare challenge in Southeast Asia. Despite advancements in screening approaches and treatment modalities, significant medical gaps remain, ranging from prevention and early diagnosis to determining targeted therapy and establishing personalized approaches to managing CRC. There is a need to expand more validated biomarkers in clinical practice. An advanced technique incorporating high-throughput mass spectrometry as a liquid biopsy to unravel a repertoire of glycoproteins and glycans would potentially drive the development of clinical tools for CRC screening, diagnosis and monitoring, and it can be further adapted to the existing standard-of-care procedure. Therefore this review offers a perspective on glycoproteomics-driven liquid biopsy and its potential integration into the clinical care of CRC in the southeast Asia region. Colorectal cancer (CRC) is a significant concern in Southeast Asia, with gaps in prevention, diagnosis and therapy. Finding glycoproteins' and glycans' potentials as CRC biomarkers could revolutionize CRC care, from screening to treatment, across the region. [ABSTRACT FROM AUTHOR]

Background: Beta-1,4-galactosyltransferase-3 (B4GALT3) belongs to the family of beta-1,4-galactosyltransferases (B4GALTs) and is responsible for the transfer of UDP-galactose to terminal N-acetylglucosamine. B4GALT3 is differentially expressed in tumors and adjacent normal tissues, and is correlated with clinical prognosis in several cancers, including neuroblastoma, cervical cancer, and bladder cancer. However, the exact role of B4GALT3 in the tumor immune microenvironment (TIME) remains unclear. Here, we aimed to elucidate the function of B4GALT3 in the TIME. Methods: To study the functions of B4GALT3 in cancer immunity, either weakly or strongly immunogenic tumor cells were subcutaneously transplanted into wild-type (WT) and B4galt3 knockout (KO) mice. Bone marrow transplantation and CD8+ T cell depletion experiments were conducted to elucidate the role of immune cells in suppressing tumor growth in B4galt3 KO mice. The cell types and gene expression in the tumor region and infiltrating CD8+ T cells were analyzed using flow cytometry and RNA sequencing. N-glycosylated proteins from WT and B4galt3 KO mice were compared using the liquid chromatography tandem mass spectrometry (LC-MS/MS)-based glycoproteomic approach. Results: B4galt3 KO mice exhibited suppressed growth of strongly immunogenic tumors with a notable increase in CD8+ T cell infiltration within tumors. Notably, B4galt3 deficiency led to changes in N-glycan modification of several proteins, including integrin alpha L (ITGAL), involved in T cell activity and proliferation. In vitro experiments suggested that B4galt3 KO CD8+ T cells were more susceptible to activation and displayed increased downstream phosphorylation of FAK linked to ITGAL. Conclusion: Our study demonstrates that B4galt3 deficiency can potentially boost anti-tumor immune responses, largely through enhancing the influx of CD8+ T cells. B4GALT3 might be suppressing cancer immunity by synthesizing the glycan structure of molecules on the CD8+ T cell surface, as evidenced by the changes in the glycan structure of ITGAL in immune cells. Importantly, B4galt3 KO mice showed no adverse effects on growth, development, or reproduction, underscoring the potential of B4GALT3 as a promising and safe therapeutic target for cancer treatment. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) represents a significant burden globally, which ranks sixth among the most frequently diagnosed cancers and stands as the third leading cause of cancer-related mortality. Glycoproteomics, as an important branch of proteomics, has already made significant achievements in the field of HCC research. Aberrant protein glycosylation has shown to promote the malignant transformation of hepatocytes by modulating a wide range of tumor-promoting signaling pathways. The glycoproteome provides valuable information for understanding cancer progression, tumor immunity, and clinical outcome, which could serve as potential diagnostic, prognostic, and therapeutic tools in HCC. In this review, recent advances of glycoproteomics contribute to clinical applications (diagnosis and prognosis) and molecular mechanisms (hepatocarcinogenesis, progression, stemness and recurrence, and drug resistance) of HCC are summarized. Glycoproteomics shows promise in HCC, enhancing early detection, risk stratification, and personalized treatments. Challenges include sample heterogeneity, diverse glycans structures, sensitivity issues, complex workflows, limited databases, and incomplete understanding of immune cell glycosylation. Addressing these limitations requires collaborative efforts, technological advancements, standardization, and validation studies. Future research should focus on targeting abnormal protein glycosylation therapeutically. Advancements in glycobiomarkers and glycosylation-targeted therapies will greatly impact HCC diagnosis, prognosis, and treatment. [ABSTRACT FROM AUTHOR]

MYC is a powerful transcription factor overexpressed in many human cancers including B cell and prostate cancers. Antibody therapeutics are exciting opportunities to attack cancers but require knowledge of surface proteins that change due to oncogene expression. To identify how MYC overexpression remodels the cell surface proteome in a cell autologous fashion and in different cell types, we investigated the impact of MYC overexpression on 800 surface proteins in three isogenic model cell lines either of B cell or prostate cell origin engineered to have high or low MYC levels. We found that MYC overexpression resulted in dramatic remodeling (both up- and down-regulation) of the cell surfaceome in a cell type-dependent fashion. We found systematic and large increases in distinct sets of >80 transporters including nucleoside transporters and nutrient transporters making cells more sensitive to toxic nucleoside analogs like cytarabine, commonly used for treating hematological cancers. Paradoxically, MYC overexpression also increased expression of surface proteins driving cell turnover such as TNFRSF10B, also known as death receptor 5, and immune cell attacking signals such as the natural killer cell activating ligand NCR3LG1, also known as B7-H6. We generated recombinant antibodies to these two targets and verified their up-regulation in MYC overexpression cell lines and showed they were sensitive to bispecific T cell engagers (BiTEs). Our studies demonstrate how MYC overexpression leads to dramatic bidirectional remodeling of the surfaceome in a cell type-dependent but functionally convergent fashion and identify surface targets or combinations thereof as possible candidates for cytotoxic metabolite or immunotherapy.

Macrophages are important immune cells that participate in both innate and adaptive immune responses, such as phagocytosis, recognition of molecular patterns, and activation of the immune response. In this study, murine peritoneal macrophages were isolated and then activated by LPS, HSV and VSV. Integrative proteomic and precision N-glycoproteomic profiling were conducted to assess the underlying macrophage activation. We identified a total of 587 glycoproteins, including 1239 glycopeptides, 526 monosaccharide components, and 8326 intact glycopeptides in glycoproteomics, as well as a total of 4496 proteins identified in proteomic analysis. These glycoproteins are widely involved in important biological processes, such as antigen presentation, cytokine production and glycosylation progression. Under the stimulation of the different pathogens, glycoproteins showed a dramatic change. We found that receptors in the Toll-like receptor pathway, such as Tlr2 and CD14, were increased under LPS and HSV stimulation. Glycosylation of those proteins was proven to influence their subcellular locations.

Immunopeptidomics, the study of peptide antigens presented on the cell surface by the major histocompatibility complex (MHC), offers insights into how our immune system recognises self/non-self in health and disease. We recently discovered that hyper-processed (remodelled) N-glycans are dominant features decorating viral spike immunopeptides presented via MHC-class II (MHC-II) molecules by dendritic cells pulsed with SARS-CoV-2 spike protein, but it remains unknown if endogenous immunopeptides also undergo N-glycan remodelling. Taking a multi-omics approach, we here interrogate published MHC-II immunopeptidomics datasets of cultured monocyte-like (THP-1) and breast cancer-derived (MDA-MB-231) cell lines for overlooked N-glycosylated peptide antigens, which we compare to their source proteins in the cellular glycoproteome using proteomics and N-glycomics data from matching cell lines. Hyper-processed chitobiose core and paucimannosidic N-glycans alongside under-processed oligomannosidic N-glycans were found to prevalently modify MHC-II-bound immunopeptides isolated from both THP-1 and MDA-MB-231, while complex/hybrid-type N-glycans were (near-)absent in the immunopeptidome as supported further by new N-glycomics data generated from isolated MHC-II-bound peptides derived from MDA-MB-231 cells. Contrastingly, the cellular proteomics and N-glycomics data from both cell lines revealed conventional N-glycosylation rich in complex/hybrid-type N-glycans, which, together with the identification of key lysosomal glycosidases, suggest that MHC-II peptide antigen processing is accompanied by extensive N-glycan trimming. N-glycan remodelling appeared particularly dramatic for cell surface-located glycoproteins while less remodelling was observed for lysosomal-resident glycoproteins. Collectively, our findings indicate that both under- and hyper-processed N-glycans are prevalent features of endogenous MHC-II immunopeptides, an observation that demands further investigation to enable a better molecular-level understanding of immune surveillance.

Glycosylation, a prevalent post-translational modification, plays a significant role in diverse biological processes and exhibits associations with various diseases, including cancer. Among the different types of glycosylation, N-glycosylation is particularly prominent. Glycosylation analysis also plays a crucial role in understanding the efficacy of protein biopharmaceuticals, as the majority of these drugs are glycoproteins. The precise detection and characterization of N-glycosylation require a series of enrichment steps. On the other hand, peptide fragmentation methods constitute a crucial step in reducing sample complexity. Moreover, enrichment strategies coupled with mass spectrometry are essential for the analysis and identification of N-glycopeptides. This research aims to enhance the detection methods for N-glycopeptides and N-glycoproteins by employing strategies that involve peptide fractionation and glycopeptide enrichment. This study targeted to assess and compare the efficacy of an integration method and a direct enrichment approach in terms of identifying glycopeptides from the glycoproteome of human plasma. The results demonstrate that the integration method detects 212 N-glycopeptides and 88 N-glycoproteins, while Cotton-HILIC direct enrichment detects 88 N-glycopeptides and 41 N-glycoproteins. In conclusion, the employment of Cotton-HILIC enrichment with high pH fractionation exhibits greater qualitative abilities in characterizing the structures and functions of glycopeptides.

BackgroundThere is a need for new serum biomarkers for early detection of hepatocellular carcinoma (HCC). Haptoglobin (Hp) N-glycosylation is altered in HCC, but the diagnostic value of site-specific Hp glycobiomarkers is rarely reported. We aimed to determine the site-specific glycosylation profile of Hp for early-stage HCC diagnosis.MethodHp glycosylation was analyzed in the plasma of patients with liver diseases (n=57; controls), early-stage HCC (n=50) and late-stage HCC (n=32). Hp phenotype was determined by immunoblotting. Hp was immunoisolated and digested into peptides. N-glycopeptides were identified and quantified using liquid chromatography–mass spectrometry. Cohort samples were compared using Wilcoxon rank-sum (Mann-Whitney U) tests. Diagnostic performance was assessed using receiver operating characteristic (ROC) curves and area under curve (AUC).ResultsSignificantly higher fucosylation, branching and sialylation of Hp glycans, and expression of high-mannose glycans, was observed as disease progressed from cirrhosis to early- and late-stage HCC. Several glycopeptides demonstrated high values for early diagnosis of HCC, with an AUC of 93% (n=1), >80% (n=3), >75% (n=13) and >70% (n=11), compared with alpha-fetoprotein (AFP; AUC of 79%). The diagnostic performance of the identified biomarkers was only slightly affected by Hp phenotype.ConclusionWe identified a panel of Hp glycopeptides that are significantly differentially regulated in early- and late-stage HCC. Some glycobiomarkers exceeded the diagnostic value of AFP (the most commonly used biomarker for HCC diagnosis). Our findings provide evidence that glycobiomarkers can be effective in the diagnosis of early HCC – individually, as a panel of glycopeptides or combined with conventional serological biomarkers.

Proteins are ubiquitously modified with glycans of varied chemical structures through distinct glycosidic linkages, making the landscape of protein glycosylation challenging to map. Profiling of intact glycopeptides with mass spectrometry (MS) has recently emerged as a powerful tool for revealing matched information of the glycosylation sites and attached glycans (i.e. intact glycosites), but is largely limited to individual glycosylation types. Herein, we describe Click‐iG, which integrates metabolic labeling of glycans with clickable unnatural sugars, an optimized MS method, and a tailored version of pGlyco3 software to enable simultaneous enrichment and profiling of three types of intact glycopeptides: N‐linked, mucin‐type O‐linked, and O‐GlcNAcylated glycopeptides. We demonstrate the utility of Click‐iG by the identification of thousands of intact glycosites in cell lines and living mice. From the mouse lung, heart, and spleen, a total of 2053 intact N‐glycosites, 262 intact O‐GalNAc glycosites, and 1947 O‐GlcNAcylation sites were identified. Click‐iG‐enabled comprehensive coverage of the protein glycosylation landscape lays the foundation for interrogating crosstalk between different glycosylation pathways. [ABSTRACT FROM AUTHOR]

The brain extracellular matrix (ECM) is a highly glycosylated environment and plays important roles in many processes including cell communication, growth factor binding, and scaffolding. The formation of structures such as perineuronal nets (PNNs) is critical in neuroprotection and neural plasticity, and the formation of molecular networks is dependent in part on glycans. The ECM is also implicated in the neuropathophysiology of disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), and Schizophrenia (SZ). As such, it is of interest to understand both the proteomic and glycomic makeup of healthy and diseased brain ECM. Further, there is a growing need for site‐specific glycoproteomic information. Over the past decade, sample preparation, mass spectrometry, and bioinformatic methods have been developed and refined to provide comprehensive information about the glycoproteome. Core ECM molecules including versican, hyaluronan and proteoglycan link proteins, and tenascin are dysregulated in AD, PD, and SZ. Glycomic changes such as differential sialylation, sulfation, and branching are also associated with neurodegeneration. A more thorough understanding of the ECM and its proteomic, glycomic, and glycoproteomic changes in brain diseases may provide pathways to new therapeutic options. [ABSTRACT FROM AUTHOR]

Congenital disorders of glycosylation (CDG) and mitochondrial disorders are multisystem disorders with overlapping symptomatology. Pathogenic variants in the PMM2 gene lead to abnormal N-linked glycosylation. This disruption in glycosylation can induce endoplasmic reticulum stress, contributing to the disease pathology. Although impaired mitochondrial dysfunction has been reported in some CDG, cellular bioenergetics has never been evaluated in detail in PMM2-CDG. This prompted us to evaluate mitochondrial function and autophagy/mitophagy in vitro in PMM2 patient-derived fibroblast lines of differing genotypes from our natural history study. We found secondary mitochondrial dysfunction in PMM2-CDG. This dysfunction was evidenced by decreased mitochondrial maximal and ATP-linked respiration, as well as decreased complex I function of the mitochondrial electron transport chain. Our study also revealed altered autophagy in PMM2-CDG patient-derived fibroblast lines. This was marked by an increased abundance of the autophagosome marker LC3-II. Additionally, changes in the abundance and glycosylation of proteins in the autophagy and mitophagy pathways further indicated dysregulation of these cellular processes. Interestingly, serum sorbitol levels (a biomarker of disease severity) and the CDG severity score showed an inverse correlation with the abundance of the autophagosome marker LC3-II. This suggests that autophagy may act as a modulator of biochemical and clinical markers of disease severity in PMM2-CDG. Overall, our research sheds light on the complex interplay between glycosylation, mitochondrial function, and autophagy/mitophagy in PMM2-CDG. Manipulating mitochondrial dysfunction and alterations in autophagy/mitophagy pathways could offer therapeutic benefits when combined with existing treatments for PMM2-CDG. [ABSTRACT FROM AUTHOR]