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Detergents assisted glycoprotein capture

ABSTRACT: A detergents assisted glycoprotein capture method was developed to increase the coverage of N-glycoproteome of various samples.Application of this approach in the larger scale N-glycoproteomics analysis of the HEK 293 cell membrane led to the identification of 2253 unique N-glycosites from 953 proteins. Application of this approach to human serum resulted in the identification of 850 N-glycosylation sites without any immuno-depletion and fractionation.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Early Embryonic Cell

SUBMITTER: Rui Chen

LAB HEAD: Daniel Figeys

PROVIDER: PXD003485 | Pride | 2016-12-23

REPOSITORIES: Pride

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			Action	DRS
	20111115_detergentsassistedglycoproteincapture_membrane_1_1.raw	Raw
	20111115_detergentsassistedglycoproteincapture_membrane_1_2.raw	Raw
	20111115_detergentsassistedglycoproteincapture_membrane_2_1.raw	Raw
	20111115_detergentsassistedglycoproteincapture_membrane_2_2.raw	Raw
	20111115_detergentsassistedglycoproteincapture_serum_1.raw	Raw

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Publications

Detergent-Assisted Glycoprotein Capture: A Versatile Tool for In-Depth N-Glycoproteome Analysis.

Chen Rui R Zou Hanfa H Figeys Daniel D

Journal of proteome research 20160512 6

Large-scale N-glycoproteome studies have been hindered by poor solubility of hydrophobic membrane proteins and the complexity of proteome samples. Herein, we developed a detergent-assisted glycoprotein capture method to reduce these issues by conducting hydrazide chemistry-based glycoprotein capture in the presence of strong detergents such as sodium dodecyl sulfate and Triton X-100. The strong detergents helped to solubilize hydrophobic membrane proteins and then increased the access of hydrazi ...[more]

PMID: 27147131

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Comprehensive analysis of platelet glycoprotein Ibα glycosylation.

Project description:Background: Platelet glycoprotein (GP) Ibα is the major ligand-binding subunit of the GPIb-IX-V complex that binds von Willebrand Factor (VWF). GPIbα is heavily glycosylated, and its glycans have been proposed to play key roles in platelet clearance, VWF binding, and as target antigens in immune thrombocytopenia syndromes. Despite its importance in platelet biology, the glycosylation profile of GPIbα is not well characterized. Objectives: The aim of this study was to comprehensively analyze GPIbα amino acid sites of glycosylation (glycosites) and glycan structures. Methods: GPIbα ectodomain that was recombinantly expressed or that was purified from human platelets was analyzed by Western blot, mass spectrometry (MS) glycomics, and MS glycoproteomics to define glycosites and the structures of the attached glycans. Results: We identified a diverse repertoire of N- and O-glycans, including sialoglycans, Tn antigen, T antigen, and ABH blood group antigens. In the analysis of the recombinant protein, we identified 62 unique O-glycosites. In the analysis of the endogenous protein purified from platelets, we identified at least 48 unique O-glycosites and 1 N-glycosite. The GPIbα mucin domain is densely O-glycosylated. Glycosites are also located within the macroglycopeptide domain and mechanosensory domain (MSD). Conclusions: This comprehensive analysis of GPIbα glycosylation lays the foundation for further studies to determine the functional and structural roles of GPIbα glycans.

2023-02-09 | PXD035030 | Pride

A magnetic bead-based workflow for sensitive and streamlined cell surface proteomics

Project description:Cell-surface proteins represent an important class of molecules for therapeutic targeting and defining cellular phenotypes. However, their enrichment and detection via mass spectrometry-based proteomics remains challenging due to low abundance, posttranslational modifications, hydrophobic regions, and processing requirements. To improve the identification of cell-surface proteins via their corresponding N-linked glycopeptides (N-glycopeptides), we optimized a Cell-Surface Capture (CSC) workflow which incorporates magnetic bead-based processing. Using this approach, we evaluated labeling conditions (biotin tags and catalysts), enrichment specificity (streptavidin beads), missed cleavages (lysis buffers), non-enzymatic deamidation (digestion and de-glycosylation buffers), and data acquisition methods. Our findings support the use of alkoxyamine-PEG4-biotin plus 5-methoxy-anthranilic acid and streptavidin magnetic beads for maximal N-glycopeptide detection. Furthermore, single-pot solid-phased-enhanced sample-preparation (SP3) circumvented the need to isolate cell membranes by affording the use of strong detergents and chaotropes for protein extraction. Notably, with semi-automated processing, sample handling was simplified and between ~600-900 N-glycoproteins were identified from only 25-200µg of HeLa protein. Overall, the improved efficiency of the magnetic-based CSC workflow allowed us to identify both previously reported and novel N-glycosites with less material and high reproducibility, and should help advance the field of surfaceomics by providing insight in cellular phenotypes not previously documented.

2024-01-21 | PXD043678 | Pride

Site-specific N-glycosylation Characterization of Recombinant SARS-CoV-2 Spike Proteins using High-Resolution Mass Spectrometry

Project description:The global pandemic of severe acute pneumonia syndrome (COVID-19) caused by SARS-CoV-2 urgently calls for prevention and intervention strategies. The densely glycosylated spike (S) protein highly exposed on the viral surface is a determinant for virus binding and invasion into host cells as well as elicitation of a protective host immune response. Herein, we characterized the site-specific N-glycosylation of SARS-CoV-2 S protein using stepped collision energy (SCE) mass spectrometry (MS). Following digestion with two complementary proteases to cover all potential N-glycosylation sequons and integrated N-glycoproteomics analysis, we revealed the N-glycosylation profile of SARS-CoV-2 S proteins at the levels of intact N-glycopeptides and glycosites, along with the glycan composition and site-specific number of glycans. All 22 potential canonical N-glycosites were identified in S protein protomer. Of those, 18 N-glycosites were conserved between SARS-CoV and SARS-CoV-2 S proteins. Nearly all glycosites were preserved among the 753 SARS-CoV-2 genome sequences available in the public influenza database Global Initiative on Sharing All Influenza Data. By comparison, insect cell-expressed SARS-CoV-2 S protein contained 38 N-glycans, which were primarily assigned to the high-mannose type N-glycans, whereas the human cell-produced protein possessed up to 140 N-glycans largely belonging to the complex type. In particular, two N-glycosites located in the structurally exposed receptor-binding domain of S protein exhibited a relatively conserved N-glycan composition in human cells. This N-glycosylation profiling and determination of differences between distinct expression systems could shed light on the infection mechanism and promote development of vaccines and targeted drugs.

2020-10-05 | PXD018506 | Pride

Sugar-assisted ligation in glycoprotein synthesis.

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N-glycoproteomic analyses from different insect species reveal conserved and unique glycoproteins

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Protein N-glycosylation is sex-related in Nilaparvata lugens adults

Project description:Nilaparvata lugens, or the brown planthopper, is one of the most notorious pest insects of cultured rice, and a model for hemimetabolous development. Recently, the N-glycome of N. lugens was explored throughout post-embryonic development and reproductive stages, which revealed differential protein N-glycosylation events between adult sexes. Identifying the proteins carrying these differential carbohydrate structures would point to the functionality of sex-dependent N-glycosylation. Furthermore, potential effects of the adult wing type on protein N-glycosylation are of interest. Here, a comprehensive investigation of the N-glycosylation sites from the adult stages of N. lugens was conducted, allowing a qualitative and quantitative comparison between sexes and wing forms at the glycopeptide level. N-glycopeptide enrichment using the N-glyco-FASP method with the high mannose/paucimannose-binding lectin Concanavalin A, or the Rhizoctonia solani agglutinin which interacts with complex N-glycans led to the identification of over 1,300 N-glycosylation sites derived from over 600 glycoproteins. Comparison of these N-glycopeptides revealed striking differences in protein N-glycosylation between sexes, while almost no differences were observed between wing types. Male- and female-specific N-glycosylation sites were identified, and some of these sex-specific N-glycosites were shown to be derived from proteins with a putative role in insect reproduction. Transcript expression experiments with complete insects and insect tissues confirmed the sex-related N-glycosylation of proteins, and expression of glycoproteins in reproductive tissues. In conclusion, this study provides original data on N-glycosylation sites of N. lugens adults, providing novel insights into planthopper’s biology and revealing that protein N-glycosylation is sex-related in this insect.

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