Title : 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