Dataset Information

GlycoIP: An Integrated Platform for the Simultaneous Profiling of Intact N- and O-glycopeptides from Human Semen

ABSTRACT: 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.

ORGANISM(S): Homo Sapiens

SUBMITTER: Yong Zhang

PROVIDER: PXD051171 | iProX | Mon Apr 01 00:00:00 BST 2024

REPOSITORIES: iProX

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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.

Dataset Information

GlycoIP: An Integrated Platform for the Simultaneous Profiling of Intact N- and O-glycopeptides from Human Semen

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