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

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.

journal_name

J Proteome Res

authors

Hong Q,Ruhaak LR,Stroble C,Parker E,Huang J,Maverakis E,Lebrilla CB

doi

10.1021/acs.jproteome.5b00756

subject

Has Abstract

pub_date

2015-12-04 00:00:00

pages

5179-92

issue

12

eissn

1535-3893

issn

1535-3907

journal_volume

14

pub_type

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