A Method for Comprehensive Glycosite-Mapping and Direct Quantitation of Serum Glycoproteins.
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.
Department of Chemistry and ‡Department of Dermatology, School of Medicine, University of California , Davis, California 95616, United States.
Qiuting
Q
Hong
Department of Chemistry and ‡Department of Dermatology, School of Medicine, University of California , Davis, California 95616, United States.
L Renee
LR
Ruhaak
Department of Chemistry and ‡Department of Dermatology, School of Medicine, University of California , Davis, California 95616, United States.
Carol
C
Stroble
Department of Chemistry and ‡Department of Dermatology, School of Medicine, University of California , Davis, California 95616, United States.
Evan
E
Parker
Department of Chemistry and ‡Department of Dermatology, School of Medicine, University of California , Davis, California 95616, United States.
Jincui
J
Huang
Department of Chemistry and ‡Department of Dermatology, School of Medicine, University of California , Davis, California 95616, United States.
Emanual
E
Maverakis
Department of Chemistry and ‡Department of Dermatology, School of Medicine, University of California , Davis, California 95616, United States.
Carlito B
CB
Lebrilla
D000595:Amino Acid Sequence; D002853:Chromatography, Liquid / Q000379:methods; D006020:Glycopeptides / Q000737:chemistry; D006023:Glycoproteins / Q000097:blood* / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D007136:Immunoglobulins / Q000097:blood / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods*; D015203:Reproducibility of Results; D000511:alpha-Macroglobulins / Q000737:chemistry
D006020:Glycopeptides; D006023:Glycoproteins; D007136:Immunoglobulins; D000511:alpha-Macroglobulins
absolute quantitation; glycopeptide; immunoglobulin; site specific glycan analysis