The effects of immortalization on the N-glycome and proteome of CDK4-transformed lung cancer cells.

Biological experiments are often conducted in vitro using immortalized cells due to their accessibility and ease of propagation compared to primary cells and live animals. However, immortalized cells may present different proteomic and glycoproteomic characteristics from the primary cell source due to the introduction of genes that enhance proliferation (e.g. CDK4) or enable telomere lengthening. To demonstrate the changes in phenotype upon CDK4-transformation, we performed LC-MS/MS glycomic and proteomic characterizations of a human lung cancer primary cell line (DTW75) and a CDK4-transformed cell line (GL01) derived from DTW75. We observed that the primary and CDK4-transformed cells expressed significantly different levels of sialylated, fucosylated, and sialofucosylated N-glycans. Specifically, the primary cells expressed higher levels of hybrid- and complex-type sialylated N-glycans, while CDK4-transformed cells expressed higher levels of complex-type fucosylated and sialofucosylated N-glycans. Further, we compared the proteomic differences between the cell lines and found that CDK4-transformed cells expressed higher levels of RNA-binding and adhesion proteins. Further, we observed that the CDK4-transformed cells changed N-glycosylation after 31 days in cell culture, with a decrease in high-mannose and increase in fucosylated, sialylated, and sialofucosylated N-glycans. Identifying these changes between primary and CDK4-transformed cells will provide useful insight when adapting cell lines that more closely resemble in vivo physiological conditions.

Integrating transcriptomics, glycomics and glycoproteomics to characterize hepatitis B virus-associated hepatocellular carcinoma.

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.

CSF N-Glycomics Using High-Throughput UPLC-ESI Techniques in Alzheimer's Disease.

In this chapter, we will present a high-throughput method applied in our laboratory for the structural elucidation of the cerebrospinal fluid (CSF) N-glycome. This methodology is based on a commercial equipment developed by WATERS™ to speed up N-deglycosylation and N-glycan labeling of glycoproteins of pharmaceutical and biological interest such as monoclonal antibodies. This analytical kit is sold under the trade name of RapiFluor-MS (RFMS). We have slightly modified the methodology, increasing the glycosylation time and using a high-resolution mass analyzer for the analysis of CSF N-glycans, thus obtaining a high-throughput method (up to 96 samples simultaneously), mass accuracy better than 5 ppm, and the ability to separate and identify isomers.

CSF N-Glycomics Using MALDI MS Techniques.

In this chapter, we will present the methodology currently applied in our laboratory for the structural elucidation of the cerebrospinal fluid (CSF) N-glycome. N-glycans are released from denatured carboxymethylated glycoproteins by digestion with peptide-N-glycosidase F (PNGase F) and purified using both C18 Sep-Pak® and porous graphitized carbon (PGC) HyperSep™ Hypercarb™ solid phase extraction (SPE) cartridges. The glycan pool is subsequently permethylated to increase mass spectrometry sensitivity. Molecular assignments are performed through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) analysis considering either the protein N-linked glycosylation pathway or MALDI TOF MS/MS data. Each stage has been optimized to obtain high-quality mass spectra in reflector mode with an optimal signal-to-noise ratio up to m/z 4800. This method has been successfully adopted to associate specific N-glycome profiles to the early and the advanced phases of Alzheimer's disease (AD).

Analysis of Native and Permethylated N-Glycan Isomers Using MGC-LC-MS Techniques.

Glycosylation is an important post-translational modification that affects many critical cellular functions such as adhesion, signaling, protein stability, and function, among others. Abnormal glycosylation has been linked to many diseases. As such, the investigation of glycans and their roles in disease pathway and progression is important. Glycan analysis can be challenging, however, due to such factors as the heterogeneity of glycans and isomers as well as the poor ionization efficiency provided by mass spectrometry analyses. This chapter presents efficient methods that overcome these and other challenges for the analysis of native and permethylated N-glycan isomers in biological samples. Instructions regarding the packing of the MGC column, the N-glycan sample prep, and the LC-MS conditions are also provided.

Targeted Glycoproteomics Analysis Using MRM/PRM Approaches.

MS-target analyses are frequently utilized to analyze and validate structural changes of biomolecules across diverse fields of study such as proteomics, glycoproteomics, glycomics, lipidomics, and metabolomics. Targeted studies are commonly conducted using multiple reaction monitoring (MRM) and parallel reaction monitoring (PRM) techniques. A reliable glycoproteomics analysis in intricate biological matrices is possible with these techniques, which streamline the analytical workflow, lower background interference, and enhance selectivity and specificity.

Current look at the most promising proteomic and glycomic biomarkers of bladder cancer.

BACKGROUND: Bladder cancer (BC) belongs to the most frequent cancer types. The diagnostic process is still long and costly, with a high percentage of false-positive or -negative results. Due to the cost and lack of effectiveness, older methods need to be supplemented or replaced by a newer more reliable method. In this regard, proteins and glycoproteins pose high potential. METHODS: We performed an online search in PubMed/Medline, Scopus, and Web of Science databases to find relevant studies published in English up until May 2023. If applicable, we set the AUC threshold to 0.90 and sensitivity/specificity (SN/SP) to 90%. FINDINGS: Protein and glycoprotein biomarkers are a demonstrably viable option in BC diagnostics. Cholinesterase shows promise in progression-free survival. BLCA-4, ORM-1 along with HTRA1 in the detection of BC. Matrix metallopeptidase 9 exhibits potential for stratification of muscle-invasive subtypes with high negative predictive value for aggressive phenotypes. Distinguishing non-muscle invasive subtypes benefits from Keratin 17. Neu5Gc-modified UMOD glycoproteins pose potential in BC diagnosis, while fibronectin, laminin-5, collagen type IV, and lamprey immunity protein in early detection of BC.

GlyComb: A novel glycoconjugate data repository that bridges glycomics and proteomics.

The glycosylation of proteins and lipids is known to be closely related to the mechanisms of various diseases such as influenza, cancer, and muscular dystrophy. Therefore, it has become clear that the analysis of post-translational modifications of proteins, including glycosylation, is important to accurately understand the functions of each protein molecule and the interactions among them. In order to conduct large-scale analyses more efficiently, it is essential to promote the accumulation, sharing, and reuse of experimental and analytical data in accordance with the FAIR (Findability, Accessibility, Interoperability, and Re-usability) data principles. However, a FAIR data repository for storing and sharing glycoconjugate information, including glycopeptides and glycoproteins, in a standardized format did not exist. Therefore, we have developed GlyComb (https://glycomb.glycosmos.org) as a new standardized data repository for glycoconjugate data. Currently, GlyComb can assign a unique identifier to a set of glycosylation information associated with a specific peptide sequence or UniProt ID. By standardizing glycoconjugate data via GlyComb identifiers and coordinating with existing web resources such as GlyTouCan and GlycoPOST, a comprehensive system for data submission and data sharing among researchers can be established. Here we introduce how GlyComb is able to integrate the variety of glycoconjugate data already registered in existing data repositories to obtain a better understanding of the available glycopeptides and glycoproteins, and their glycosylation patterns. We also explain how this system can serve as a foundation for a better understanding of glycan function.

Integrated analysis of natural glycans using a versatile pyrazolone-type heterobifunctional tag ANPMP.

Glycans mediate various biological processes through carbohydrate-protein interactions, and glycan microarrays have become indispensable tools for understanding these mechanisms. However, advances in functional glycomics are hindered by the absence of convenient and universal methods for obtaining natural glycan libraries with diverse structures from glycoconjugates. To address this challenge, we have developed an integrative approach that enables one-pot release and simultaneously capture, separation, structural characterization, and functional analysis of N/O-glycans. Using this approach, glycoconjugates are incubated with a pyrazolone-type heterobifunctional tag-ANPMP to obtain glycan-2ANPMP conjugates, which are then converted to glycan-AEPMP conjugates. We prepared a tagged glycan library from porcine gastric mucin, soy protein, human milk oligosaccharides, etc. Following derivatization by N-acetylation and permethylation, glycans were subjected to detailed structural characterization by ESI-MSn analysis, which revealed >83 highly pure glycan-AEPMPs containing various natural glycan epitopes. A shotgun microarray is constructed to study the fine details of glycan-bindings by proteins and antisera.

Systematic review: Glycomics as diagnostic markers for hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most prevalent primary liver cancer with one of the highest cancer-related mortality rates worldwide. Early diagnosis is crucial for improving the therapeutic options and reducing the disease-related mortality. AIM: To investigate serum N-glycomics as diagnostic markers for HCC. METHODS: We performed a comprehensive search in PubMed, EMBASE, Web of Science and Scopus through August 17, 2023. Eligible studies assessed the potential use of serum N-glycomics as diagnostic biomarkers for HCC. Study selection, data extraction and quality assessment were performed by two independent reviewers. RESULTS: Of the 48 articles included, 11 evaluated the utility of N-glycomics for the diagnosis of HCC in whole serum while the remaining articles focused on specific protein glycoforms or protein levels. Of these specific proteins, haptoglobin, alpha-fetoprotein (AFP), kininogen (Kin), α-1-antitrypsin and Golgi protein 73 (GP73) were the most frequently studied. Increased levels of fucosylation and branching presented as the most prevalent post-translational modifications of glycoproteins in patients with HCC compared to controls. Notably, glycomics-based biomarkers may provide a clinical benefit for the diagnosis of early HCC, as several algorithms achieved AUCs between 0.92-0.97. However, these were based on single studies with limited sample sizes and should therefore be validated. CONCLUSIONS: Alterations in serum N-glycomics, characterised by increased levels of fucosylation and branching, have potential as diagnostic biomarkers for HCC. Optimisation of study design, patient selection and analysing techniques are needed before clinical implementation will be possible.

Comprehensive Comparison of Bioactive N-Glycans among Seven Species of Livestock and Poultry Plasma Using a Relative Quantification Strategy.

Glycans have been proven to play special roles in keeping human health as a class of nutritional and bioactive ingredients in many food materials. However, their broad use in the food industry is hindered by the lack of comprehensive analytical methods for high-quality food glycomics studies and large-quantity raw materials for their production. This study focuses on structural identification and quantitative comparison of bioactive N-glycans in seven species of livestock and poultry plasma as potential natural glycan resources by a novel comprehensive relative quantification strategy based on stable isotope labeling with nondeuterated and deuterated 4-methyl-1-(2-hydrazino-2-oxoethyl)-pyridinium bromide (d0/d7-HMP) in combination with linkage-specific derivatization of sialic acid residues. Methodological validation of the method in terms of detection sensitivity, signal resolution, quantification linearity, precision, and accuracy on model neutral and complicated sialylated glycans demonstrated its advantages over the existing methods. Based on this method, a series of bioactive N-glycans were found in seven species of livestock and poultry plasma, and their differences in structure, abundance percentages, and relative contents of N-glycans were revealed, demonstrating their excellent applicability for comprehensive food glycomics analysis and great exploitation potential of these plasma samples as large-quantity raw materials in producing bioactive N-glycans for application in food and pharmaceutical industries.

Glycosyltransferases as versatile tools to study the biology of glycans.

All cells are decorated with complex carbohydrate structures called glycans that serve as ligands for glycan-binding proteins (GBPs) to mediate a wide range of biological processes. Understanding the specific functions of glycans is key to advancing an understanding of human health and disease. However, the lack of convenient and accessible tools to study glycan-based interactions has been a defining challenge in glycobiology. Thus, the development of chemical and biochemical strategies to address these limitations has been a rapidly growing area of research. In this review, we describe the use of glycosyltransferases (GTs) as versatile tools to facilitate a greater understanding of the biological roles of glycans. We highlight key examples of how GTs have streamlined the preparation of well-defined complex glycan structures through chemoenzymatic synthesis, with an emphasis on synthetic strategies allowing for site- and branch-specific display of glyco-epitopes. We also describe how GTs have facilitated expansion of glyco-engineering strategies, on both glycoproteins and cell surfaces. Coupled with advancements in bioorthogonal chemistry, GTs have enabled selective glyco-epitope editing of glycoproteins and cells, selective glycan subclass labeling, and the introduction of novel biomolecule functionalities onto cells, including defined oligosaccharides, antibodies, and other proteins. Collectively, these approaches have contributed great insight into the fundamental biological roles of glycans and are enabling their application in drug development and cellular therapies, leaving the field poised for rapid expansion.

Decoding glycomics with a suite of methods for differential expression analysis.

Glycomics, the comprehensive profiling of all glycan structures in samples, is rapidly expanding to enable insights into physiology and disease mechanisms. However, glycan structure complexity and glycomics data interpretation present challenges, especially for differential expression analysis. Here, we present a framework for differential glycomics expression analysis. Our methodology encompasses specialized and domain-informed methods for data normalization and imputation, glycan motif extraction and quantification, differential expression analysis, motif enrichment analysis, time series analysis, and meta-analytic capabilities, synthesizing results across multiple studies. All methods are integrated into our open-source glycowork package, facilitating performant workflows and user-friendly access. We demonstrate these methods using dedicated simulations and glycomics datasets of N-, O-, lipid-linked, and free glycans. Differential expression tests here focus on human datasets and cancer vs. healthy tissue comparisons. Our rigorous approach allows for robust, reliable, and comprehensive differential expression analyses in glycomics, contributing to advancing glycomics research and its translation to clinical and diagnostic applications.