Dataset Information

Improving quantitative glycoproteomics by systematic evaluation of different glycopeptide enrichment, quantification, and mass spectrometry strategies

ABSTRACT: In this study, we conducted a systematic investigation into a glycoproteomic workflow. Firstly, Label-free and TMT labeling strategies were utilized for intact N-glycopeptide quantification. A total of 2,924 unique intact N-glycopeptides can be quantified after being labeled using TMT approach. Compared to label-free method, TMT labeling for its high precision with the CV averaging 8% and multiplex analysis capabilities. Subsequently, we compared the capacity of ZIC-HILIC and MAX enrichment methods, revealing that the ZIC- HILIC method demonstrated superior performance in enriching intact N-glycopeptides, which can identify 3,004 GPSMs from a single injection of patient-derived xenograft (PDX) sample. Furthermore, we systematically evaluated different higher-energy collisional dissociation (HCD) collision energies, determining that the stepped collision energy HCD (sceHCD) settings of 25, 35, and 45 was the optimal for both the identification and quantification of intact N-glycopeptides.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Breast

SUBMITTER: Zhenyu Sun

LAB HEAD: Hui Zhang

PROVIDER: PXD054874 | Pride | 2025-05-07

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	02PDX_P9697_P_JHU_20231129_Ascend_F01.raw	Raw
	02PDX_P9697_P_JHU_20231129_Ascend_F02.raw	Raw
	02PDX_P9697_P_JHU_20231129_Ascend_F03.raw	Raw
	02PDX_P9697_P_JHU_20231129_Ascend_F04.raw	Raw
	02PDX_P9697_P_JHU_20231129_Ascend_F05.raw	Raw

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Publications

Improving Glycoproteomic Analysis Workflow by Systematic Evaluation of Glycopeptide Enrichment, Quantification, Mass Spectrometry Approach, and Data Analysis Strategies.

Sun Zhenyu Z Lih T Mamie TM Woo Jongmin J Jiao Liyuan L Hu Yingwei Y Wang Yuefan Y Liu Hongyi H Zhang Hui H

Analytical chemistry 20241216 52

Glycosylation is one of the most prevalent and crucial protein modifications. Quantitative site-specific characterization of glycosylation usually requires sophisticated intact glycopeptide analysis using glycoproteomics. Recent efforts have focused on the interrogation of intact glycopeptide analyses using tandem mass spectrometry. However, a systematic evaluation of the quantitative glycoproteomic workflow is still lacking. This study compared different strategies for glycopeptide enrichment a ...[more]

PMID: 39679613

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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.