Dataset Information

Site-specific glycoform analysis of human AGP and HL-60 cell lysate by the Glyco-RIDGE method using GRable Version 1.0

ABSTRACT: High-throughput intact glycopeptide analysis is crucial for elucidating the physiological and pathological status of the glycans attached to each glycoprotein. Mass spectrometry-based glycoproteomic methods are challenging because of the diversity and heterogeneity of glycan structures. Therefore, we developed an MS1-based site-specific glycoform analysis method named "Glycan heterogeneity-based Relational IDentification of Glycopeptide signals on Elution profile (Glyco-RIDGE)" for a more comprehensive analysis. This method detects glycopeptide signals as a cluster based on the mass and chromatographic properties of glycopeptides and then searches for each combination of core peptides and glycan compositions by matching their mass and retention time differences. Here, we developed a novel browser-based software named GRable for semi-automated Glyco-RIDGE analysis with significant improvements in glycopeptide detection algorithms, including "parallel clustering." This unique function improved the comprehensiveness of glycopeptide detection and allowed the analysis to focus on specific glycan structures, such as pauci-mannose. The other notable improvement is evaluating the "confidence level" of the GRable results, especially using MS2 information. This function facilitated reduced misassignment of the core peptide and glycan composition and improved the interpretation of the results. Additional improved points of the algorithms are "correction function" for accurate monoisotopic peak picking; one-to-one correspondence of clusters and core peptides even for multiply sialylated glycopeptides; and "inter-cluster analysis" function for understanding the reason for detected but unmatched clusters. The significance of these improvements was demonstrated using purified and crude glycoprotein samples, showing that GRable allowed site-specific glycoform analysis of intact sialylated glycoproteins on a large-scale and in-depth. Therefore, this software will help us analyze the status and changes in glycans to obtain biological and clinical insights into protein glycosylation by complementing the comprehensiveness of MS2-based glycoproteomics. GRable can be freely run online using a web browser via the GlyCosmos Portal (https://glycosmos.org/grable).

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Chiaki Nagai-Okatani

PROVIDER: PXD046226 | JPOST Repository | Thu Aug 29 00:00:00 BST 2024

REPOSITORIES: jPOST

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Dataset's files

Source:

			Action	DRS
	HL-60_Output-file_GRable.xlsx	Xlsx
	HL-60_Output-file_Mascot(full).xlsx	Xlsx
	HL-60_Output-file_Mascot(semi).xlsx	Xlsx
	HL-60_TL+AT+Am%232-1p4-sp-mono+dd2HCD-5-25-35-140g-01.mgf	Mgf
	HL-60_TL+AT+Am%232-1p4-sp-mono+dd2HCD-5-25-35-140g-01.mzML	Mzml

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Publications

GRable Version 1.0: A Software Tool for Site-Specific Glycoform Analysis With Improved MS1-Based Glycopeptide Detection With Parallel Clustering and Confidence Evaluation With MS2 Information.

Nagai-Okatani Chiaki C Tominaga Daisuke D Tomioka Azusa A Sakaue Hiroaki H Goda Norio N Ko Shigeru S Kuno Atsushi A Kaji Hiroyuki H

Molecular & cellular proteomics : MCP 20240823 9

High-throughput intact glycopeptide analysis is crucial for elucidating the physiological and pathological status of the glycans attached to each glycoprotein. Mass spectrometry-based glycoproteomic methods are challenging because of the diversity and heterogeneity of glycan structures. Therefore, we developed an MS1-based site-specific glycoform analysis method named "Glycan heterogeneity-based Relational IDentification of Glycopeptide signals on Elution profile (Glyco-RIDGE)" for a more compre ...[more]

PMID: 39181535

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Project description:In collaboration with Professor Anne Dell at the South Kensington Campus of Imperial College London, we have conducted preliminary glycomic surveys of C57BL/6 CD25+ and CD25- CD4+ T cells. Cells selected in serum-containing culture medium demonstrated unusual N-glycan profiles compared to those prepared in phosphate buffered saline (PBS). These profiles were subsequently shown to result from cellular sequestration of glycoproteins present in fetal calf serum (FCS) used to supplement the medium (1). Analysis of the N-glycan profile of murine CD4+CD25+ Tregs prepared in PBS revealed distinct differences from CD4+CD25- T cells – namely, a reduction in sialic acid capping and loss of core fucose. Preliminary studies are underway to ascertain the minimum medium required to sustain general T cell survival, whilst maintaining ‘clean’ glycomic profiles. The effect of glycoprotein withdrawal on CD25+ and CD25- CD4+ T cells in culture was investigated by selecting the cells in FCS-free medium and incubating them for six hours with either FCS-containing medium, or PBS supplemented with FCS, fetuin or fetuin-derived glycans. Early apoptosis was measured using Annexin-V (BD Biosciences, Heidelberg, Germany) and late apoptosis using 7-aminoactinomycin D (7-AAD) (BD Biosciences), in parallel with a complementary apoptosis detection method – measurement of the mitochondrial membrane potential using the BD™ Mitoscreen Kit (BD Biosciences). This apoptosis staining study demonstrated that there was a remarkably higher percentage of death in both CD25+ and CD25- CD4+ T cells cultured in PBS versus cells cultured in RPMI-1640 or medium containing FCS. Both CD25+ and CD25- CD4+ T cells were highly susceptible to apoptosis in glycoprotein-free medium, demonstrating their exquisite sensitivity to the lack of glycoprotein survival factors. Continuing studies are addressing the minimum medium required to sustain cells undergoing polyclonal activation in vitro and the impact of activation on the N-glycan profiles of Tregs versus conventional T cells. To complement this work, we used Core E resources to investigate glycosyltransferase gene expression in freshly isolated versus activated C57BL/6 CD25+ and CD25- CD4+ T cells.