The studies aimed towards glycan biomarker discovery have focused on glycan characterization and profiling of released glycans. Site-specific glycosylation analysis is less developed but may provide a new type 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. The remarkable sensitivity and selectivity of MRM have enabled the detection of low abundant glycopeptides from serum directly.
The glycosylation pattern of protein standards was first determined using both trypsin and pronase digestion. Proteins were treated with DTT and IAA before an overnight digestion in a 37°C water bath. Glycopeptides were profiled using an Agilent 6520 HPLC-Chip/QTOF MS, and identified using an in-house-software tool, GPFinder. Once the site specific glycosylation was determined, quantification was performed with the tryptic peptides using an Agilent 6490 QqQ MS coupled with an Agilent 1290 UPLC system. The absolute amount of proteins was determined by the peak area of peptides, while the degree of glycosylation was normalized to the protein content, thus allowing quantification of glycoforms on the site-specific and protein-specific level. The method was applied to glycoproteins in 11 healthy human sera.
We determined the site-specific glycosylation of immunoglobulin G, A, and M, transferrin, alpha-2-macroglobulin, and haptoglobin using nonspecific proteolysis. Tryptic digestion yielded limited information compared to nonspecific protease analysis, but was necessary to obtain a consistent peptide sequence for MRM.
As an example, a peptide that is common to IgG1, IgG2, IgG3 and IgG4 was quantified to obtain the absolute amount of IgG protein. The result showed a wide dynamic range (>1000) and low LOD (6.01fmole). The average IgG concentration for a set of 11 samples was 5.25mg/ml, with a relative standard deviation (RSD) of 21.8%. Peptides that were unique for each subclass were quantified to monitor the variations in abundances for the four IgG subclasses.