Lectin-based SP3 technology enables N-glycoproteomics analysis
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion Lumos
ORGANISM(S): Mus Musculus (mouse)
TISSUE(S): Testis, Oocyte
SUBMITTER: 
Yueshuai Guo
LAB HEAD: Xuejiang Guo
PROVIDER: PXD047610 | Pride | 2024-04-10
REPOSITORIES: Pride
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Project description:Glycosylation of proteins is one of the most common post-translational modification (PTM) and plays important regulatory functions in diverse biological processes such as protein stability or cell signaling. Accordingly, glycoproteins are also a consistent part of the human tear film proteome maintaining the proper function of the ocular surface and forming the first defense barrier of the ocular immune system. Irregularities in the glycoproteomic composition of the tear film might promote development of chronic eye diseases indicating glycoproteins as valuable source for biomarker discovery or drug target identification. The present study aimed to develop a lectin-based affinity method for the enrichment and concentration of tear glycoproteins/glycopeptides and the characterization of their specific N-glycosylation sites by high-resolution mass spectrometry (MS). For method development and evaluation, we accumulated first native glycoproteins from human tear sample pools and assessed the enrichment efficiency of different lectin column systems by 1D gel electrophoresis and specific protein stainings (Coomassie and glycoproteins). The best-performing multi-lectin column system (comprising the four lectins ConA, JAC, WGA and UEA I, termed as 4L) was applied for glycopeptide enrichment from human tear sample digests followed by MS-based detection and localization of their specific N-glycosylation sites. As main result, the present study identified in total 26 N glycosylation sites of 11 N-glycoproteins in tear sample pools of healthy individuals (n=3 biological sample pools). Amongst others, we identified tear film proteins lactotransferrin (N497 and N642, LTF), Ig heavy chain constant α-1 (N144 and 340, IGHA1), prolactin-inducible protein (N105, PIP) as well as extracellular lacritin (N105, LACRT) as highly reliable and significant N glycoproteins, which were already associated with the pathogenesis of various chronic eye diseases such as the dry eye syndrome (DES). In conclusion, the results of the present study will serve as important tear film N-glycoprotein catalogue for future studies focusing the human tear film and ocular surface-related inflammatory diseases.
2023-01-25 | PXD038280 | Pride
Project description:Objectives: Hashimoto’s thyroiditis (HT) is one of the most common autoimmune disorders; however, its underlying pathological mechanisms remain unclear. Although aberrant glycosylation has been implicated in the N-glycome of immunoglobulin G (IgG), changes in serum proteins have not been comprehensively characterized. This study aimed to investigate the glycosylation profile of serum samples of HT patients that were depleted of highly abundant proteins by and propose the potential functions of glycoproteins for the further study of pathological mechanisms of HT . Methods: A lectin microarray containing 70 lectins was used to detect and analyze glycosylation of serum proteins using serum samples (N=27 HT; N=26 healthy control [HC]) depleted of abundant proteins. Significant differences in glycosylation status between HT patients and the HC group were verified by lectin blot analysis. A lectin-based pull-down assay combined with mass spectrometry was used to investigate potential glycoproteins combined with differentially present lectins, and an enzyme-linked immunosorbent assay (ELISA) was used to identify the expression of targeted glycoproteins in 131 patients with papillary thyroid carcinoma (PTC), 131 patients with benign thyroid nodules (BTN) patients, 130 patients with HT, and 128 HCs. Results: Compared with the HC group, the majority of the lectin binding signals in HT group were weakened, while the Vicia villosa agglutinin (VVA) binding signal was increased. The difference in VVA binding signals verified by lectin blotting was consistent with the results of the lectin microarray. A total of 113 potential VVA-binding glycoproteins were identified by mass spectrometry and classified by gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) analyses . Using ELISA, we confirmed that lactoferrin (LTF) and mannan-binding lectin-associated serine protease 1 (MASP-1) levels were elevated in the serum of patients with HT and PTC. Conclusions: Following depletion of abundant proteins, remaining serum proteins in HT patients exhibited lower glycosylation levels than those observed in HCs. An increased level of potential VVA-binding glycoproteins may play an important role in HT development. LTF and MASP-1 expression was significantly higher in the serum of HT and PTC patients, suggesting key roles played by glycosylation in pathological mechanisms underlying HT and PTC.
2023-06-22 | PXD040847 | Pride
Project description:Dr. Esko's laboratory focuses on the structure, function, and biosynthesis of glycoproteins and proteoglycans. This laboratory also works on the design and synthesis of small molecule inhibitors of glycosylation.
2011-02-17 | GSE27351 | GEO
Project description:Tn antigen (Tn), a single N-acetylgalactosamine (GalNAc) monosaccharide attached to protein Ser and Thr residues, is found on most solid tumors yet rarely detected in adult tissues: featuring it one of the most distinctive signatures of cancers. Although it is prevalent in cancers, Tn-glycosylation sites are not entirely clear owing to the lack of suitable technology. Knowing the Tn-glycosylation sites will spur the development of the new vaccines, diagnostics, and therapeutics of cancers. Here, we report a novel technology named EXoO-Tn for large-scale mapping of Tn-glycosylation sites. EXoO-Tn utilizes glycosyltransferase C1GalT1 and isotopically-labeled UDP-Gal(13C6) to tag and convert Tn to Gal(13C6)-Tn. This exquisite Gal(13C6)-Tn structure is recognized by a human-gut-bacterial enzyme, called OpeRATOR, that specifically cleaves N-termini of the Gal(13C6)-Tn-occupied Ser and Thr residues to yield site-containing glycopeptides. The enzymes C1GalT1 and OpeRATOR could be used concurrently in one-pot. The effectiveness of EXoO-Tn was evaluated by analyzing Jurkat cells, where 947 Tn-glycosylation sites from 480 glycoproteins were mapped. Bioinformatic analysis of the identified site-specific Tn-glycoproteins revealed conserved motif, cellular localization, relative position in proteins, and mapped site-specific Tn-glycoproteome in different studies. Given the significance of Tn in cancers, EXoO-Tn is anticipated to have broad utilities in clinical study of cancers.
2022-02-15 | PXD014390 | Pride
Project description:Protein glycosylation is ubiquitous and plays critical roles in biology. However, study of O-linked glycoproteome (O-glycoproteome), a major type of protein glycosylation, has been severely impeded due to paucity of technology. We presented a chemoenzymatic strategy for extraction of site-specific O-linked glycopeptides (SOGO), which enabled simultaneous enrichment and identification of O-linked glycosylation sites (O-glycosites) in an unprecedented depth. Central to SOGO is a tandem-use of solid-phase capture of peptides and an O-linked glycan (O-glycan) dependent endoprotease named OpeRATOR. Interestingly, OpeRATOR requires O-glycan to specifically cleave the N-terminus of glycan-occupied Ser and Thr leading to unambiguous identification of O-glycosites and corresponding glycoproteins. The identified O-glycosites facilitated downstream determination of microheterogeneity of intact O-linked glycopeptides (O-glycopeptides). We benchmarked the method using human serum and identified 805 peptides containing 777 O-glycosites from 1,345 site-specific O-glycopeptides and 302 glycoproteins. We applied the method to study change of protein-specific O-glycosylation in human T cells infected with human immunodeficiency virus (HIV-1). Strikingly, 1404 peptides containing 1,352 O-glycosites from 577 glycoproteins were identified. In addition, altered site-specific O-linked glycosylation (O-glycosylation) during HIV infection of T cells were observed. In total, 1989 O-glycosites from 789 glycoproteins were precisely pinpointed from human serum and T cells. We observed conserved motifs for O-glycoproteome. Finally, ontology analysis revealed diverse roles for O-glycoproteins arguing broad application of our method to study biology in respective to protein O-glycosylation.
2022-02-28 | PXD007895 | Pride
Project description:Dr. Esko's laboratory focuses on the structure, function, and biosynthesis of glycoproteins and proteoglycans. This laboratory also works on the design and synthesis of small molecule inhibitors of glycosylation. We analyzed the expression patterns of glycosyltransferase, growth factors, and receptors in endothelial cells derived from NDST1 deficient and wild-type mice using cancer cell line types U937 and LS180 .
2011-02-17 | E-GEOD-27351 | biostudies-arrayexpress
Project description:Glycoproteins isolated from elongating cotton fiber cells were analyzed using 2D-PAGE followed by MALDI TOF/TOF based protein identification. Protein isoforms were identified using Tandem mass spectrometry. Lectin based Glycopeptide enrichment strategy followed by PNGase catalyzed deglycosylation reaction was employed to assign potential N-linked glycosylation sites.
2015-11-25 | PXD002849 | Pride
Project description:A new strategy to compare cellular glycoproteomes, thereby identifying membrane proteins with altered glycan structures and the concerned glycosites. The workflow consists of membrane proteins digestion followed by lectin-based isolation of glycopeptides and their fractionation. Since alterations in the glycan part of a glycopeptide causes mass alterations, analytical size exclusion chromatography is applied to detect these mass shifts. A combination of N-glycosidase treatment with nanoUPLC coupled Exploris-Orbitrap mass spectrometry identifies the altered glycoproteins and the respective glycosites. The methodology was established using the human colon cancer cell line CX1 which was treated with 2-deoxy-glucose - a modulator of N-glycosylation.
2021-09-10 | PXD024770 | Pride
Project description:A number of plasma glycoproteins are clinically useful as biomarkers in a variety of diseases. Although thousands of proteins are present in plasma, >95% of the plasma proteome by mass is represented by only 22 proteins. This necessitates strategies to deplete the abundant proteins and enrich other subsets of proteins. Although glycoproteins are abundant in plasma, in routine proteomic analyses, glycopeptides are not often investigated. Traditional methods such as lectin-based enrichment of glycopeptides followed by deglycosylation have helped understand the glycoproteome, but they lack any information about the attached glycans. Here, we apply size-exclusion chromatography (SEC) as a simple strategy to enrich intact glycopeptides based on their larger size which achieves broad selectivity regardless of the nature of attached glycans. Using this approach, we identified 1,317 glycopeptides belonging to 266 glycosylation sites on 154 plasma glycoproteins. The deep coverage achieved by this approach was evidenced by extensive heterogeneity that was observed. For instance, 20-100 glycopeptides were observed per protein for the top 15 glycoproteins. Overall, we found 615 novel glycopeptides of which 39 glycosylation sites (from 38 glycoproteins) are not including in protein databases such as Uniprot and GlyconnectDB. We also identified 12 novel glycopeptides containing di-sialic acid, which is a rare glycan epitope. Thus, SEC allows for efficient LC-MS/MS-based deep glycoproteomics from low amounts (~1 l) of human plasma. Overall, the SEC-based method described here is a simple, rapid and high-throughput strategy for characterization of the glycoproteome.
2021-08-18 | PXD025414 | Pride
Project description:Our studies provide direct evidence that O-glycosylation pathways play a role in the regulation of cell growth through apoptosis and proliferation pathways. Eight small molecular weight analogues of the GalNAc-alpha-1-O-serine/threonine structure based on 1-benzyl-2-acetamido-2- deoxy-alpha-O-D-galactopyranoside have been synthesised and tested in 5 human colorectal cancer cell lines. Three inhibitors, 1-benzyl-2-acetamido-2-deoxy-alpha-O-D-galactopyranoside and the corresponding 2-azido- and C-glycoside analogues, were screened in two colorectal cancer cell lines at 0.5mM and showed induction of apoptosis. Proliferation was down regulated in the same two cell lines with all three inhibitors, as detected by Ki67 staining and gene array. Treatment both cell lines with inhibitors led to changes in glycosylation detected with peanut lectin. The competitive action of the inhibitors resulted in the intracellular formation of 28 aryl-glycan products which were identified by MALDI and electrospray mass spectroscopy. The structures found map onto known O-glycosylation biosynthetic pathways and showed a differential pattern for each of the inhibitors in both cell lines. Gene array analysis of the glycogenes illustrated a pattern of glycosytransferases that matched the glycan structures found in glycoproteins and aryl-glycans formed in the PC/AA/C1/SB10C cells, however there was no action of the three inhibitors on glycogene transcript levels. The inhibitors act at both intermediary metabolic and genomic levels, resulting in altered protein glycosylation and arylglycan formation. These events may play a part in growth arrest. Keywords: Response to inhibitors, Apoptosis, Aryl-glycans, Benzyl-O-GalNAc, Growth Inhibition, O-Glycans
2008-08-18 | GSE12422 | GEO