期刊
JOURNAL OF PROTEOME RESEARCH
卷 11, 期 3, 页码 1728-1740出版社
AMER CHEMICAL SOC
DOI: 10.1021/pr201183w
关键词
mass spectrometry; HILIC chromatography; Q-TOF; haptoglobin; hemopexin; GlycoPeptideSearch software; detached glycans; oxonium ions
资金
- CCSG [NIH P30 CA51008]
- National Science Foundation
- Ministry of Education, Youth and Sports of the Czech Republic [LC545]
- [RO1 CA115625]
- [CA135069]
Glycoproteins fulfill many indispensable biological functions, and changes in protein glycosylation have been observed in various diseases. Improved analytical methods are needed to allow a complete characterization of this complex and common post-translational modification. In this study, we present a workflow for the analysis of the microheterogeneity of N-glycoproteins that couples hydrophilic interaction and nanoreverse-phase C18 chromatography to tandem QTOF mass spectrometric analysis. A glycan database search program, GlycoPeptideSearch, was developed to match N-glycopeptide MS/MS spectra with the glycopeptides comprised of a glycan drawn from the GlycomeDB glycan structure database and a peptide from a user-specified set of potentially glycosylated peptides. Application of the workflow to human haptoglobin and hemopexin, two microheterogeneous N-glycoproteins, identified a total of 57 distinct site-specific glycoforms in the case of haptoglobin and 14 site-specific glycoforms of hemopexin. Using glycan oxonium ions and peptide-characteristic glycopeptide fragment ions and by collapsing topologically redundant glycans, the search software was able to make unique N-glycopeptide assignments for 51% of assigned spectra, with the remaining assignments primarily representing isobaric topological rearrangements. The optimized workflow, coupled with GlycoPeptideSearch, is expected to make high-throughput semiautomated glycopeptide identification feasible for a wide range of users.
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