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Large-scale Identification of N-linked Intact Glycopeptides in Human Serum using HILIC Enrichment and Spectral Library Search.

ABSTRACT: Large-scale identification of N-linked intact glycopeptides by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in human serum is challenging because of the wide dynamic range of serum protein abundances, the lack of a complete serum N-glycan database and the existence of proteoforms. In this regard, a spectral library search method was presented for the identification of N-linked intact glycopeptides from N-linked glycoproteins in human serum with target-decoy and motif-specific false discovery rate (FDR) control. Serum proteins were firstly separated into low-abundance and high-abundance proteins by acetonitrile (ACN) precipitation. After digestion, the N-linked intact glycopeptides were enriched by hydrophilic interaction liquid chromatography (HILIC) and a portion of the enriched N-linked intact glycopeptides were processed by Peptide-N-Glycosidase F (PNGase F) to generate N-linked deglycopeptides. Both N-linked intact glycopeptides and deglycopeptides were analyzed by LC-MS/MS. From N-linked deglycopeptides data sets, 764 N-linked glycoproteins, 1699 N-linked glycosites and 3328 unique N-linked deglycopeptides were identified. Four types of N-linked glycosylation motifs (NXS/T/C/V, X≠P) were used to recognize the N-linked deglycopeptides. The spectra of these N-linked deglycopeptides were utilized for N-linked deglycopeptides library construction and identification of N-linked intact glycopeptides. A database containing 739 N-glycan masses was constructed and utilized during spectral library search for the identification of N-linked intact glycopeptides. In total, 526 N-linked glycoproteins, 1036 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.

SUBMITTER: Shu Q

PROVIDER: S-EPMC7124471 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Glycoprotein changes occur in not only protein abundance but also the occupancy of each glycosylation site by different glycoforms during biological or pathological processes. Recent advances in mass spectrometry instrumentation and techniques have facilitated analysis of intact glycopeptides in complex biological samples by allowing the users to generate spectra of intact glycopeptides with glycans attached to each specific glycosylation site. However, assigning these spectra, leading to identification of the glycopeptides, is challenging. Here, we report an algorithm, named GPQuest, for site-specific identification of intact glycopeptides using higher-energy collisional dissociation (HCD) fragmentation of complex samples. In this algorithm, a spectral library of glycosite-containing peptides in the sample was built by analyzing the isolated glycosite-containing peptides using HCD LC-MS/MS. Spectra of intact glycopeptides were selected by using glycan oxonium ions as signature ions for glycopeptide spectra. These oxonium-ion-containing spectra were then compared with the spectral library generated from glycosite-containing peptides, resulting in assignment of each intact glycopeptide MS/MS spectrum to a specific glycosite-containing peptide. The glycan occupying each glycosite was determined by matching the mass difference between the precursor ion of intact glycopeptide and the glycosite-containing peptide to a glycan database. Using GPQuest, we analyzed LC-MS/MS spectra of protein extracts from prostate tumor LNCaP cells. Without enrichment of glycopeptides from global tryptic peptides and at a false discovery rate of 1%, 1008 glycan-containing MS/MS spectra were assigned to 769 unique intact N-linked glycopeptides, representing 344 N-linked glycosites with 57 different N-glycans. Spectral library matching using GPQuest assigns the HCD LC-MS/MS generated spectra of intact glycopeptides in an automated and high-throughput manner. Additionally, spectral library matching gives the user the possibility of identifying novel or modified glycans on specific glycosites that might be missing from the predetermined glycan databases.

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.

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Large-scale Identification of N-linked Intact Glycopeptides in Human Serum using HILIC Enrichment and Spectral Library Search.

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