A Method for Comprehensive Glycosite-Mapping and Direct Quantitation of Serum Glycoproteins

Qiuting Hong, L. Renee Ruhaak, Carol Stroble, Evan Parker, Jincui Huang, Emanual Michael Maverakis, Carlito B Lebrilla

Research output: Contribution to journalArticle

42 Scopus citations

Abstract

A comprehensive glycan map was constructed for the top eight abundant glycoproteins in plasma using both specific and nonspecific enzyme digestions followed by nano liquid chromatography (LC)-chip/quadrupole time-of-flight mass spectrometry (MS) analysis. Glycopeptides were identified using an in-house software tool, GPFinder. A sensitive and reproducible multiple reaction monitoring (MRM) technique on a triple quadrupole MS was developed and applied to quantify immunoglobulins G, A, M, and their site-specific glycans simultaneously and directly from human serum/plasma without protein enrichments. A total of 64 glycopeptides and 15 peptides were monitored for IgG, IgA, and IgM in a 20 min ultra high performance (UP)LC gradient. The absolute protein contents were quantified using peptide calibration curves. The glycopeptide ion abundances were normalized to the respective protein abundances to separate protein glycosylation from protein expression. This technique yields higher method reproducibility and less sample loss when compared with the quantitation method that involves protein enrichments. The absolute protein quantitation has a wide linear range (3-4 orders of magnitude) and low limit of quantitation (femtomole level). This rapid and robust quantitation technique, which provides quantitative information for both proteins and glycosylation, will further facilitate disease biomarker discoveries.

Original languageEnglish (US)
Pages (from-to)5179-5192
Number of pages14
JournalJournal of Proteome Research
Volume14
Issue number12
DOIs
StatePublished - Dec 4 2015

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Keywords

  • absolute quantitation
  • glycopeptide
  • immunoglobulin
  • site specific glycan analysis

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

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