Project description:Large-scale identification of N-linked intact glycopeptides by liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) in human serum is challenging due to the wide dynamic range of serum protein abundances, the lack of a complete serum N-Glycan database and the existence of non-specifically digested peptides and numerous modifications. In this regard, a spectral library search method was presented for serum N-linked intact glycopeptides identification with target-decoy and motif-specific false discovery rate (FDR) control. Low-abundant glycoproteins were firstly separated from high-abundant proteins by acetonitrile (ACN) precipitation. After digestion, the N-linked intact glycopeptides were enriched by hydrophilic interaction liquid chromatography (HILIC) column and a portion of the enriched N-linked intact glycopeptides were processed by N-Glycosidase F (PNGase F) to generate de-glycopeptides. Both N-linked intact glycopeptides and de-glycopeptides were analyzed by LC-MS/MS. From N-linked de-glycopeptides datasets, 764 N-linked glycoproteins, 1,699 N-linked glycosites and 3,328 unique N-linked de-glycopeptides were identified. The spectra of these N-linked de-glycopeptides were utilized for N-linked de-glycopeptides library construction and identification of N-linked intact glycopeptides. Four types of N-linked glycosylation motifs (NXS/T/C/V, X≠P) were used to recognize the N-linked de-glycopeptides, and two different N-Glycan mass databases including 27 modified N-Glycan masses and 712 unmodified N-Glycan masses were combined and utilized during spectral library search for identification of N-linked intact glycopeptides. In total, from the N-linked intact glycopeptides datasets, 526 N-linked glycoproteins, 1,036 N-linked glycosites, 22,677 N-linked intact glycopeptides and 738 N-Glycan masses were identified under 1% FDR, representing the most in-depth serum N-glycoproteome identified by LC-MS/MS at N-linked intact glycopeptide level and N-linked de-glycopeptides level.
Project description:A workflow for differential analysis of the microheterogeneity of site-specific intact N-glycopeptides of serum haptoglobin between early hepatocellular carcinoma (HCC) and liver cirrhosis has been developed.
Project description:This dataset reports a comprehensive N-glycoproteomic profiling of serum-derived humanized antibodies expressed in transgenic mice using an intact glycopeptide (IGP)–based LC–MS/MS workflow. The study aimed to identify and characterize site-specific N-glycosylation modifications of mouse serum proteins, with emphasis on antibody-related glycoforms. Following sample digestion and HILIC enrichment, glycopeptides were analyzed by high-resolution Orbitrap Astral mass spectrometry in data-dependent acquisition (DDA) mode. The identified N-linked glycopeptides were annotated for glycan composition, glycosylation heterogeneity, and functional classification through GO, KEGG, Reactome, and STRING analyses. The dataset provides an extensive resource for understanding host-dependent glycosylation patterns and their potential impact on antibody functionality.
Project description:Serum N-linked intact glycopeptides was investigated in AFP-negative HCC and cirrhosis (LC) patients by using label-free quantification methodology.
Project description:The heterogeneity and low abundance of protein glycosylation present challenging barriers to the analysis of intact glycopeptides, which is key to comprehensively understanding the role of glycosylation in an organism. Efficient and specific enrichment of intact glycopeptides could help greatly with this problem. Here, we propose a new enrichment strategy using a boronic acid functionalized mesoporous graphene silica composite for isolating intact glycopeptides from complex biological samples. The merits of this composite, including high surface area and synergistic effect from size exclusion functionality of mesoporous material, hydrophilic interaction of silica, and the reversible covalent binding with BA, enable the effective and specific enrichment of both intact glycopeptides. The results from the enrichment performance of the strategy evaluated by standard glycoproteins and the application to global glycosylation analyses in human serum indicate the robustness and potential of the strategy for intact glycopeptide analysis.
Project description:We present a mass spectrometry-based glycoproteomics method that employs sequential treatment of intact glycopeptides with enzymes (STAGE) to simultaneously analyze site-specific core fucosylation and N-linked glycosylation of glycoproteins.
Project description:Protein glycosylation in human semen is crucial for spermatogenesis, maturation, sperm motility, capacitation, and fertilization. However, the site-specific information on N- and O-glycosylation in human semen has not been fully and accurately identified, particularly O-glycosylation. To address this, an integrated platform (termed GlycoIP) was developed for the simultaneous profiling of intact N- and O-glycopeptides from human semen. Although characterizing intact glycopeptides presents a challenge, their analysis can provide rich information about both the glycans and glycosites simultaneously. In this study, a total of 1,833 unique intact N-glycopeptides and 720 unique intact O-glycopeptides were reported based on GlycoIP. Much deeper and more accurate site-specific N/O-glycosylation information was revealed in this study, including 438 O-glycosites from 148 distinctive O-glycoproteins that may play important roles in human semen composition and function. In summary, GlycoIP provided a potentially useful method for the systematically profiling protein N/O-glycosylation in a single experiment. This lays the foundation for the functional studies of glycoproteins in male infertility.
Project description:Polyprenol reductase is an enzyme encoded by the SRD5A3 gene, which catalyzes the synthesis of dolichol from polyprenols. Dolichol serves as a carrier for glycan precursors in N-linked glycosylation or monosaccharides in O-glycosylation, C-mannosylation, and GPI anchor biosynthesis. Pathogenic variants in SRD5A3 can result in a congenital disorder of glycosylation (CDG), SRD5A3-CDG, which is inherited in an autosomal recessive manner. Most serum proteins undergo glycosylation and changes in the glycosylation levels of numerous serum glycoproteins have been associated with pathological consequences. Despite the critical role of SRD5A3 in glycosylation, the impact of its deficiency on the glycosylation of serum proteins remains largely unexplored. In this study, we used tandem mass tags (TMT)-based multiplexed quantitation approach to analyze serum N-glycoproteins and proteins in SRD5A3-CDG patients and controls. We quantified 2,200 serum N-glycopeptides from 359 N-glycosites from 204 serum proteins. Extensive hypoglycosylation of serum proteins was observed in patients, with 245 of 291 dysregulated glycopeptides showing hypoglycosylation. These significantly changing glycopeptides belonged to several known abundant serum glycoproteins including haptoglobin, plasma serine protease inhibitor, alpha-1-B glycoprotein, alpha-2-macroglobulin and ceruloplasmin. We also detected changes in glycopeptides from albumin which has recently been shown to be glycosylated. Overall, our study provides novel insights into alterations in the glycosylation status of serum proteins in SRD5A3-CDG that could facilitate further investigations into diagnostics and therapeutics aspects of this CDG.
Project description:Protein glycosylation is one of the most common protein modifications and plays essential roles in biology and therapeutics. However, the analysis of in vivo O-linked glycosylation, a major type of protein glycosylation, has been severely impeded by the scarcity of technology. Here, a chemoenzymatic method was presented for the site-specific extraction of O-linked glycopeptides (EXoO), which achieved simultaneous enrichment and unambiguous mapping of over 3,000 O-linked glycosylation sites and corresponding O-linked glycans on over 1,000 proteins in human kidney tissues, serum and T cells. The large-scale localization of O-linked glycosylation sites nearly doubles the sites identified in the last decades demonstrating that EXoO is the most effective method to-date for defining the site-specific O-linked glycoproteome in different types of sample. Structural analysis of the sites revealed conserved motifs and topological orientation facing extracellular space or lumen of ER and Golgi. Striking signature of aberrant in vivo O-linked glycoproteome was observed between kidney tumor and normal tissues discovering key factors in tumor biology. The O-linked glycoproteome play diverse roles on the ER, Golgi membrane, cell surface and extracellular space arguing that EXoO can be applied broadly to the analysis of O-linked glycoproteins in biology and therapeutics.