The use of multiple reaction monitoring on QQQ-MS for the analysis of protein- and site-specific glycosylation patterns in serum

Abstract

In recent years, high-throughput glycomics approaches have been developed and applied to either complete biofl uids, cell lysates or tissues, or proteins isolated thereof. However, during such analyses the N-glycan are released from the protein backbone and therefore site- and protein-specifi c information is lost. There exists a need for high-throughput methods that allow quantifi cation of site- and protein- specifi c glycosylation patterns from complex biological mixtures. We here describe the use of a multiple reaction monitoring mass spectrometry based method for the generation of glycopeptide profi les of the nine high abundance glycoproteins IgG, IgA, IgM, haptoglobin, alpha-1-antitrypsin, alpha-2- macroglobulin, alpha-1-acid glycoprotein, transferrin, and complement C3. We show that the sample preparation can be performed at the 96-well level, and using a 17-min gradient on a RP-UPLC-QQQ instrument, 96 samples can be analyzed within 3 days.