title: Direct glycan structure determination of intact N-linked glycopeptides by low-energy collision-induced dissociation tandem mass spectrometry and predicted spectral library searching.
abstract: Intact glycopeptide MS analysis to reveal site-specific protein glycosylation is an important frontier of proteomics. However, computational tools for analyzing MS/MS spectra of intact glycopeptides are still limited and not well-integrated into existing workflows. In this work, a new computational tool which combines the spectral library building/searching tool, SpectraST (Lam et al. Nat. Methods2008, 5, 873-875), and the glycopeptide fragmentation prediction tool, MassAnalyzer (Zhang et al. Anal. Chem.2010, 82, 10194-10202) for intact glycopeptide analysis has been developed. Specifically, this tool enables the determination of the glycan structure directly from low-energy collision-induced dissociation (CID) spectra of intact glycopeptides. Given a list of possible glycopeptide sequences as input, a sample-specific spectral library of MassAnalyzer-predicted spectra is built using SpectraST. Glycan identification from CID spectra is achieved by spectral library searching against this library, in which both m/z and intensity information of the possible fragmentation ions are taken into consideration for improved accuracy. We validated our method using a standard glycoprotein, human transferrin, and evaluated its potential to be used in site-specific glycosylation profiling of glycoprotein datasets from LC-MS/MS. In addition, we further applied our method to reveal, for the first time, the site-specific N-glycosylation profile of recombinant human acetylcholinesterase expressed in HEK293 cells. For maximum usability, SpectraST is developed as part of the Trans-Proteomic Pipeline (TPP), a freely available and open-source software suite for MS data analysis.
authors: Department of Chemical and Biomolecular Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong; Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; Center for Infectious Disease and Cancer Research, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors: Pei-Jing
authors: PJ
authors: Pai
authors: Department of Chemical and Biomolecular Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong.
authors: Yingwei
authors: Y
authors: Hu
authors: Department of Chemical and Biomolecular Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong; Division of Biomedical Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong. Electronic address: kehlam@ust.hk.
authors: Henry
authors: H
authors: Lam
mesh_terms: D006020:Glycopeptides / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D019151:Peptide Library; D011134:Polysaccharides / Q000032:analysis*; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism
chemical_list: D006020:Glycopeptides; D019151:Peptide Library; D011134:Polysaccharides; D014357:Trypsin
keywords: Low-energy collision-induced dissociation tandem mass spectrometry; N-glycopeptide identification; Predicted spectral library searching; Site-specific glycosylation analysis