Absolute quantitation of immunoglobulin G and its glycoforms using multiple reaction monitoring.
Studies aimed toward glycan biomarker discovery have focused on glycan characterization by the global profiling of released glycans. Site-specific glycosylation analysis is less developed but may provide new types of biomarkers with higher sensitivity and specificity. Quantitation of peptide-conjugated glycans directly facilitates the differential analysis of distinct glycoforms associated with specific proteins at distinct sites. We have developed a method using MRM to monitor protein glycosylation normalized to absolute protein concentrations to examine quantitative changes in glycosylation at a site-specific level. This new approach provides information regarding both the absolute amount of protein and the site-specific glycosylation profile and will thus be useful to determine if altered glycosylation profiles in serum/plasma are due to a change in protein glycosylation or a change in protein concentration. The remarkable sensitivity and selectivity of MRM enable the detection of low abundance IgG glycopeptides, even when IgG was digested directly in serum with no cleanup prior to the liquid chromatography. Our results show a low limit of detection of 60 amol and a wide dynamic range of 3 orders magnitude for IgG protein quantitation. The results show that IgG glycopeptides can be analyzed directly from serum (without enrichment) and yield more accurate abundances when normalized to the protein content. This report represents the most comprehensive study so far of the use of multiple reaction monitoring for the quantitation of glycoproteins and their glycosylation patterns in biofluids.
Department of Chemistry, University of California , One Shields Avenue, Davis, California, 95616, United States.
Qiuting
Q
Hong
Carlito B
CB
Lebrilla
Suzanne
S
Miyamoto
L Renee
LR
Ruhaak
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D007074:Immunoglobulin G