Glycopeptide Profiling Research Articles

15N metabolic labeling-TMT multiplexing approach to facilitate the quantitation of glycopeptides derived from cell lines.

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 15N 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 14N and 15N 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.

Click-iG: Simultaneous Enrichment and Profiling of Intact N-linked, O-GalNAc, and O-GlcNAcylated Glycopeptides.

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.

Click‐iG: Simultaneous Enrichment and Profiling of Intact N‐linked, O‐GalNAc, and O‐GlcNAcylated Glycopeptides

AbstractProteins 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.

Roles of glycosylation at the cancer cell surface: opportunities for large scale glycoproteomics.

Cell surface glycosylation has a variety of functions, and its dysregulation in cancer contributes to impaired signaling, metastasis and the evasion of the immune responses. Recently, a number of glycosyltransferases that lead to altered glycosylation have been linked to reduced anti-tumor immune responses: B3GNT3, which is implicated in PD-L1 glycosylation in triple negative breast cancer, FUT8, through fucosylation of B7H3, and B3GNT2, which confers cancer resistance to T cell cytotoxicity. Given the increased appreciation of the relevance of protein glycosylation, there is a critical need for the development of methods that allow for an unbiased interrogation of cell surface glycosylation status. Here we provide an overview of the broad changes in glycosylation at the surface of cancer cell and describe selected examples of receptors with aberrant glycosylation leading to functional changes, with emphasis on immune checkpoint inhibitors, growth-promoting and growth-arresting receptors. Finally, we posit that the field of glycoproteomics has matured to an extent where large-scale profiling of intact glycopeptides from the cell surface is feasible and is poised for discovery of new actionable targets against cancer.

Profiling Serum Intact N-Glycopeptides Using Data-Independent Acquisition Mass Spectrometry.

Large-scale profiling of intact glycopeptides is critical but challenging in glycoproteomics. Data-independent acquisition (DIA) mass spectrometry is an emerging technology with deep proteome coverage as well as accurate quantitative capability for large-scale proteomics studies and has also been applied to the field of glycoproteomics. In this protocol, we describe how to analyze data from a DIA experiment for profiling serum intact N-glycopeptides. We present a comprehensive data analysis workflow using GproDIA, including glycopeptide spectral library building, chromatographic feature extraction from the DIA data, and feature scoring with appropriate statistical control of error rates. We anticipate that this method could provide a powerful tool to explore the serum glycoproteome.

Abstract 2799: Peripheral blood glycoproteomic biomarkers as a powerful new tool for the detection of nasopharyngeal carcinoma

Abstract Introduction: Nasopharyngeal carcinoma (NPC) is a malignancy characterized by distinct geographic, ethnic, and genetic differences. It presents a major threat to human health, particularly in East Asia and Southeast Asia. NPC commonly invades adjacent tissues and organs, resulting in complex and difficult to treat clinical presentations. NPC commonly remains largely asymptomatic during early disease stages due to its small primary foci. Hence, NPC patients are diagnosed mostly at advanced stage. Currently, no accurate and effective methods are available for the early diagnosis, evaluation, or prognosis for NPC. While Epstein-Barr virus (EBV) DNA and EBV serology tests have been advocated for early diagnosis and screening for NPC, the sensitivities of these two tests vary. due to the fact that only a small proportion of individuals infected with EBV ultimately develop NPC. The ability to detect neoplasms at early stages provides useful information for early intervention. We have recently demonstrated that interrogating the serum glycoproteome, using a proprietary platform that couples artificial intelligence (AI) to targeted liquid chromatography-massspectrometry (LC-MS) yields highly informative biomarkers in a range of neoplastic diseases. Experimental procedures in this proof-of-principle study, a total of 106 plasma samples (1:1 ratio of histologically confirmed cases of NPC and cancer-free controls), which were matched for age (average 49 years) were used. All subjects were male, from a multi-ethnic patient population in Malaysia, including three ethnic minority groups (Bidayuh, Iban and Kadaza). To identify diagnostic glycoprotein profiles for NPC we applied an in-house developed targeted glycoproteomics workflow, whereby plasma proteins were first digested with trypsin/LysC, followed by targeted analysis using LC-MS. The LC-MS generated chromatographic peaks were integrated using an in-house AI-based software for high throughput analysis and further subjected to multivariate statistical analysis. Summary of new data among the 579 glycopeptides interrogated in this study we found 62 glycopeptides statistically significantly abundant in NPC cases compared to healthy controls (at FDR <0.1). From these, we built a predictive algorithm that differentiates NPC cases from controls with high sensitivity and selectivity (AUC of 0.955). Six glycopeptide markers were found as associated with ethnicity. Conclusions: In summary, our results indicate that LC-MS-based glycopeptide profiling provides a highly accurate means of for differentiating NPC cases from healthy controls. Follow-up studies with larger sample size are currently in progress. Citation Format: Klaus Lindpaintner, Thi Thin Aye, Gege Xu, Rachel Rice, Alan Mtchell, Daniel Serie, A Malek, LH Low, Lp Tan, Ct Low, Cy Wong, H Sawali, Yy Yapp, As Khoo. Peripheral blood glycoproteomic biomarkers as a powerful new tool for the detection of nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2799.

Selective reaction monitoring approach using structure-defined synthetic glycopeptides for validating glycopeptide biomarkers pre-determined by bottom-up glycoproteomics.

Clusterin is a heavily glycosylated protein that is upregulated in various cancer and neurological diseases. The findings by the Hancock and Iliopoulos group that levels of the tryptic glycopeptide derived from plasma clusterin, 372Leu-Ala-Asn-Leu-Thr-Gln-Gly-Glu-Asp-Gln-Tyr-Tyr-Leu-Arg385 with a biantennary disialyl N-glycan (A2G2S2 or FA2G2S2) at Asn374 differed significantly prior to and after curative nephrectomy for clear cell renal cell carcinoma (RCC) patients motivated us to verify the feasibility of this glycopeptide as a novel biomarker of RCC. To determine the precise N-glycan structure attached to Asn374, whether A2G2S2 is composed of the Neu5Acα2,3Gal or/and the Neu5Acα2,6Gal moiety, we synthesized key glycopeptides having one of the two putative isomers. Selective reaction monitoring assay using synthetic glycopeptides as calibration standards allowed “top-down glycopeptidomics” for the absolute quantitation of targeted label-free glycopeptides in a range from 313.3 to 697.5 nM in the complex tryptic digests derived from serum samples of RCC patients and healthy controls. Our results provided evidence that the Asn374 residue of human clusterin is modified dominantly with the Neu5Acα2,6Gal structure and the levels of clusterin bearing an A2G2S2 with homo Neu5Acα2,6Gal terminals at Asn374 decrease significantly in RCC patients as compared with healthy controls. The present study elicits that a new strategy integrating the bottom-up glycoproteomics with top-down glycopeptidomics using structure-defined synthetic glycopeptides enables the confident identification and quantitation of the glycopeptide targets pre-determined by the existing methods for intact glycopeptide profiling.

GproDIA enables data-independent acquisition glycoproteomics with comprehensive statistical control

Large-scale profiling of intact glycopeptides is critical but challenging in glycoproteomics. Data independent acquisition (DIA) is an emerging technology with deep proteome coverage and accurate quantitative capability in proteomics studies, but is still in the early stage of development in the field of glycoproteomics. We propose GproDIA, a framework for the proteome-wide characterization of intact glycopeptides from DIA data with comprehensive statistical control by a 2-dimentional false discovery rate approach and a glycoform inference algorithm, enabling accurate identification of intact glycopeptides using wide isolation windows. We further utilize a semi-empirical spectrum prediction strategy to expand the coverage of spectral libraries of glycopeptides. We benchmark our method for N-glycopeptide profiling on DIA data of yeast and human serum samples, demonstrating that DIA with GproDIA outperforms the data-dependent acquisition-based methods for glycoproteomics in terms of capacity and data completeness of identification, as well as accuracy and precision of quantification. We expect that this work can provide a powerful tool for glycoproteomic studies.

Benefits of Chemical Sugar Modifications Introduced by Click Chemistry for Glycoproteomic Analyses.

Mucin-type O-glycosylation is among the most complex post-translational modifications. Despite mediating many physiological processes, O-glycosylation remains understudied compared to other modifications, simply because the right analytical tools are lacking. In particular, analysis of intact O-glycopeptides by mass spectrometry is challenging for several reasons; O-glycosylation lacks a consensus motif, glycopeptides have low charge density which impairs ETD fragmentation, and the glycan structures modifying the peptides are unpredictable. Recently, we introduced chemically modified monosaccharide analogs that allowed selective tracking and characterization of mucin-type O-glycans after bioorthogonal derivatization with biotin-based enrichment handles. In doing so, we realized that the chemical modifications used in these studies have additional benefits that allow for improved analysis by tandem mass spectrometry. In this work, we built on this discovery by generating a series of new GalNAc analog glycopeptides. We characterized the mass spectrometric signatures of these modified glycopeptides and their signature residues left by bioorthogonal enrichment reagents. Our data indicate that chemical methods for glycopeptide profiling offer opportunities to optimize attributes such as increased charge state, higher charge density, and predictable fragmentation behavior.

Hyper-truncated Asn355- and Asn391-glycans modulate the activity of neutrophil granule myeloperoxidase

Myeloperoxidase (MPO) plays essential roles in neutrophil-mediated immunity via the generation of reactive oxidation products. Complex carbohydrates decorate MPO at discrete sites, but their functional relevance remains elusive. To this end, we have characterised the structure–biosynthesis–activity relationship of neutrophil MPO (nMPO). Mass spectrometry demonstrated that nMPO carries both characteristic under-processed and hyper-truncated glycans. Occlusion of the Asn355/Asn391-glycosylation sites and the Asn323-/Asn483-glycans, located in the MPO dimerisation zone, was found to affect the local glycan processing, thereby providing a molecular basis of the site-specific nMPO glycosylation. Native mass spectrometry, mass photometry and glycopeptide profiling revealed significant molecular complexity of diprotomeric nMPO arising from heterogeneous glycosylation, oxidation, chlorination and polypeptide truncation variants and a previously unreported low-abundance monoprotomer. Longitudinal profiling of maturing, mature, granule-separated and pathogen-stimulated neutrophils demonstrated that nMPO is dynamically expressed during granulopoiesis, unevenly distributed across granules and degranulated upon activation. We also show that proMPO-to-MPO maturation occurs during early/mid-stage granulopoiesis. While similar global MPO glycosylation was observed across conditions, the conserved Asn355-/Asn391-sites displayed elevated glycan hyper-truncation, which correlated with higher enzyme activities of MPO in distinct granule populations. Enzymatic trimming of the Asn355-/Asn391-glycans recapitulated the activity gain and showed that nMPO carrying hyper-truncated glycans at these positions exhibits increased thermal stability, polypeptide accessibility and ceruloplasmin-mediated inhibition potential relative to native nMPO. Finally, molecular modelling revealed that hyper-truncated Asn355-glycans positioned in the MPO-ceruloplasmin interface are critical for uninterrupted inhibition. Here, through an innovative and comprehensive approach, we report novel functional roles of MPO glycans, providing new insight into neutrophil-mediated immunity.

Mass Spectrometry-Based Methods for Immunoglobulin G N-Glycosylation Analysis.

Mass spectrometry and its hyphenated techniques enabled by the improvements in liquid chromatography, capillary electrophoresis, novel ionization, and fragmentation modes are truly a cornerstone of robust and reliable protein glycosylation analysis. Boost in immunoglobulin G (IgG) glycan and glycopeptide profiling demands for both applied biomedical and research applications has brought many new advances in the field in terms of technical innovations, sample preparation, improved throughput, and confidence in glycan structural characterization. This chapter summarizes mass spectrometry basics, focusing on IgG and monoclonal antibody N-glycosylation analysis on several complexity levels. Different approaches, including antibody enrichment, glycan release, labeling, and glycopeptide preparation and purification, are covered and illustrated with recent breakthroughs and examples from the literature omitting excessive theoretical frameworks. Finally, selected highly popular methodologies in IgG glycoanalytics such as liquid chromatography-mass spectrometry and matrix-assisted laser desorption ionization are discussed more thoroughly yet in simple terms making this text a practical starting point either for the beginner in the field or an experienced clinician trying to make sense out of the IgG glycomic or glycoproteomic dataset.

High-throughput glycopeptide profiling of prostate-specific antigen from seminal plasma by MALDI-MS

An altered total seminal plasma glycosylation has been associated with male infertility, and the highly abundant seminal plasma glycoprotein prostate-specific antigen (PSA) plays an important role in fertilization. However, the exact role of PSA glycosylation in male fertility is not clear. To understand the involvement of PSA glycosylation in the fertilization process, analytical methods are required to study the glycosylation of PSA from seminal plasma with a high glycoform resolution and in a protein-specific manner. In this study, we developed a novel, high-throughput PSA glycopeptide workflow, based on matrix-assisted laser desorption/ionization-mass spectrometry, allowing the discrimination of sialic acid linkage isomers via the derivatization of glycopeptides. The method was successfully applied on a cohort consisting of seminal plasma from infertile and fertile men (N = 102). Forty-four glycopeptides were quantified in all samples, showing mainly complex-type glycans with high levels of fucosylation and sialylation. In addition, N,N-diacetyllactosamine (LacdiNAc) motives were found as well as hybrid-type and high mannose-type structures. Our method showed a high intra- and interday repeatability and revealed no difference in PSA glycosylation between fertile and infertile men. Next to seminal plasma, the method is also expected to be of use for studying PSA glycopeptides derived from other biofluids and/or in other disease contexts.

Simultaneous Immunoglobulin A and G Glycopeptide Profiling for High-Throughput Applications.

Immunoglobulin (Ig) glycosylation is recognized for its influence on Ig turnover and effector functions. However, the large-scale profiling of Ig glycosylation in a biomedical setting is challenged by the existence of different Ig isotypes and subclasses, their varying serum concentrations, and the presence of multiple glycosylation sites per Ig. Here, a high-throughput nanoliquid chromatography (LC)- mass spectrometry (MS)-based method for simultaneous analysis of IgG and IgA glycopeptides was developed and applied on a serum sample set from 185 healthy donors. Sample preparation from minute amounts of serum was performed in 96-well plate format. Prior to trypsin digestion, IgG and IgA were enriched simultaneously, followed by a one-step denaturation, reduction, and alkylation. The obtained nanoLC-MS data were subjected to semiautomated, targeted feature integration and quality control. The combined and simplified protocol displayed high overall method repeatability, as assessed using pooled plasma and serum standards. Taking all samples together, 143 individual N- and O-glycopeptides were reliably quantified. These glycopeptides were attributable to 11 different peptide backbones, derived from IgG1, IgG2/3, IgG4, IgA1, IgA2, and the joining chain from dimeric IgA. Using this method, novel associations were found between IgA N- and O-glycosylation and age. Furthermore, previously reported associations of IgG Fc glycosylation with age in healthy individuals were confirmed. In conclusion, the new method paves the way for high-throughput multiprotein plasma glycoproteomics.

Profiling of Endogenously Intact N-Linked and O-Linked Glycopeptides from Human Serum Using an Integrated Platform

Endogenous glycopeptides in serum are an invaluable resource for biomarker discovery. Due to the low abundance and the poor fragmentation in tandem mass spectrometry, the identification of endogenously intact glycopeptides still faces many challenges. Herein, an integrated platform is fabricated for the identification of N-linked and O-linked endogenously intact glycopeptides. In this platform, the high-temperature acid denaturation, ultrafiltration, and hydrophilic interaction chromatography steps are combined together for the highly efficient extraction of the endogenously intact glycopeptides from a small amount of serum. Additionally, the twin-spectra scheme and in silico deglycosylation strategy were applied for the identification of N-linked and O-linked endogenous glycopeptides, respectively. In total, 223 intact N-glycopeptides and 51 intact O-glycopeptides are identified from only 40 μL of the human serum sample. This is the first study reporting the identification of endogenously intact N-linked and O-linked glycopeptide and is also the largest data set of endogenously intact glycopeptides reported so far. The distributions of glycans among peptides and proteins and cleavage sites on peptides are further analyzed to seek the regulation of endogenous glycosylation for disease mechanism. The developed strategy provides a novel platform for the disease biomarker discovery.

Glycosylation of serum haptoglobin as a marker of gastric cancer: an overview for clinicians

ABSTRACTIntroduction: Gastric cancer (GC) is one of the leading causes of cancer-related death worldwide because of difficulties in early diagnosis. Aberrant glycosylation in serum proteins has been associated with many human diseases. Serum haptoglobin, a highly sialylated glycoprotein with four N-glycosylation sites, has gained considerable attention due to its potential as a signature molecule to display aberrant glycosylation in inflammatory disorders and various types of cancer. In particular, the relevance of haptoglobin glycosylation in GC has been investigated in a multifaceted way.Areas covered: The screening of haptoglobin glycosylation could offer an alternative approach toward GC diagnosis and detection. In this report, various assay platforms such as glycan profiling, site-specific glycopeptide profiling, and intact protein profiling are introduced for the detection of abnormal glycosylation of serum haptoglobin.Expert opinion: Although aberrant glycosylation of serum haptoglobin is associated with gastric cancer patients and might be a promising marker of GC screening, the development of a diagnosis platform to increase specificity and sensitivity for clinical use is still an analytical challenge. However, the continuous advancement of analytical technologies and methods will spur the paradigm shift from traditional serum markers, enabling the effective mining of human glycoproteome for GC diagnostic markers.

Dual-Functional Titanium(IV) Immobilized Metal Affinity Chromatography Approach for Enabling Large-Scale Profiling of Protein Mannose-6-Phosphate Glycosylation and Revealing Its Predominant Substrates.

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

An integrated approach for comprehensive profiling and quantitation of IgG-Fc glycopeptides with application to rheumatoid arthritis

Glycosylation plays an important role in the maintenance of the structure and function of glycoproteins, while aberrant protein glycosylation is correlated with various diseases. Human immunoglobulin G (IgG), which is composed of four subclasses (IgG1, IgG2, IgG3 and IgG4), is one of the most dominant and significant glycoprotein in human serum. The glycosylation on IgG-Fc moiety is known to be alternated with various physiological and pathological states. We herein report an integrated approach for comprehensive profiling and quantitation of IgG-Fc glycopeptides. Firstly, IgG N-glycans were profiled by using mAb-Glyco chip quadrupole time-of-flight mass spectrometry (Q-TOF-MS), resulting characterization of 87 N‑glycans originating from 29 different oligosaccharide compositions. Secondly, subclass-specific glycopeptides were identified on the basis of high-resolution MS and MS/MS data by using ultra high performance liquid chromatography (UHPLC) coupled to Q-TOF-MS. As a result, 83 IgG-Fc glycopeptides from human serum, including 17 sialylated glycopeptides, were identified. In addition, a quantitation method with high sensitivity and repeatability was established by using UHPLC triple quadrupole (QQQ) MS. We applied this approach to carry out quantitative analysis of IgG glycosylation in RA patients. Finally, 36 potential glycopeptide biomarkers, including 13 species from IgG1, 12 species from IgG2/3 and 11 species from IgG4 were identified.

Capturing site-specific heterogeneity with large-scale N-glycoproteome analysis

Protein glycosylation is a highly important, yet poorly understood protein post-translational modification. Thousands of possible glycan structures and compositions create potential for tremendous site heterogeneity. A lack of suitable analytical methods for large-scale analyses of intact glycopeptides has limited our abilities both to address the degree of heterogeneity across the glycoproteome and to understand how this contributes biologically to complex systems. Here we show that N-glycoproteome site-specific microheterogeneity can be captured via large-scale glycopeptide profiling methods enabled by activated ion electron transfer dissociation (AI-ETD), ultimately characterizing 1,545 N-glycosites (>5,600 unique N-glycopeptides) from mouse brain tissue. Our data reveal that N-glycosylation profiles can differ between subcellular regions and structural domains and that N-glycosite heterogeneity manifests in several different forms, including dramatic differences in glycosites on the same protein. Moreover, we use this large-scale glycoproteomic dataset to develop several visualizations that will prove useful for analyzing intact glycopeptides in future studies.

High Anti-Interfering Profiling of Endogenous Glycopeptides for Human Plasma by the Dual-Hydrophilic Metal-Organic Framework.

Glycopeptidome profiling provides large-scale information about the glycosylation level of endogenous peptides, reflecting the dynamic processes of disease occurrences and developments. However, endogenous glycopeptides are usually submerged in complex fluids containing a wide variety of interference molecules, such as high concentration proteins, nonglycopeptides, and salts, which confounds attempts to identify glycopeptidome. Here, a dual-hydrophilic metal-organic framework is developed to selectively capture endogenous glycopeptides in complex biological fluid. The hydrophilic matrix material provides specific selectivity toward glycopeptides, while the deliberate surface regulation using hydrophilic species enhances its interaction with glycopeptides. This hydrophilic probe presents an extremely high performance in anti-interfering enrichment of glycopeptides from mimic complex samples, even when the molar ratio of immunoglobulin G versus bovine serum albumin was up to about 1:5000. More excitingly, in the practical application of glycopeptidome analysis, a total of 380 endogenous N-glycopeptides with 180 unique N-glycopeptide sites were identified from human plasma. This strategy is expected to broaden the application of dual-hydrophilic MOF-based materials, especially in dealing with the challenges of extremely complex biological samples.

Evidence of Altered Glycosylation of Serum Proteins Prior to Pancreatic Cancer Diagnosis.

Biomarkers for the early detection of pancreatic cancer are urgently needed. The aim of this pilot study was to evaluate changes in serum N-glycoproteins and their glycosylation status prior to clinical presentation of pancreatic cancer that may be potential biomarkers. Prediagnosis serum samples pooled according to five time-to-diagnosis groups and a non-cancer control pool were digested with trypsin, labelled with mass tags, and subjected to titanium dioxide capture, deglycosylation, and 2D-LC-MS/MS profiling. Unbound peptides were profiled in parallel. Across the sample groups, 703 proteins were quantified and 426 putative sites of N-glycosylation were identified with evidence of several novel sites. Altered proteins with biomarker potential were predominantly abundant inflammatory response, coagulation, and immune-related proteins. Whilst glycopeptide profiles largely paralleled those of their parent proteins, there was evidence of altered N-glycosylation site occupancy or sialic acid content prior to diagnosis for some proteins, most notably of immunoglobulin gamma chains. α-1-Antitrypsin was tested as a biomarker, but found not to complement carbohydrate antigen 19-9 (CA19-9) in early detection of cancer. In conclusion, we provide preliminary evidence of altered glycosylation of several serum proteins prior to pancreatic cancer diagnosis, warranting further investigation of these proteins as early biomarkers. These changes may be largely driven by inflammatory processes that occur in response to tumour formation and progression.