The Use of Multiple Reaction Monitoring on QQQ-MS for the Analysis of Protein- and Site-Specific Glycosylation Patterns in Serum

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 1503; p. 63
• Main Author: Ruhaak, L Renee
• Format: Journal Article
• Language: English
• Published: United States 01.01.2017
• Subjects: Blood Proteins - analysis; Blood Proteins - chemistry; Glycopeptides - blood; Glycopeptides - chemistry; Glycoproteins - blood; Glycoproteins - chemistry; Glycosylation; Humans; Immunoglobulin Isotypes - blood; Immunoglobulin Isotypes - chemistry; Mass Spectrometry - methods; Serum; Quantitation; N-glycopeptides; Multiple reaction monitoring; RP-LC-MS
• DOI: 10.1007/978-1-4939-6493-2_6

In recent years, high-throughput glycomics approaches have been developed and applied to either complete biofluids, 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-specific information is lost. There exists a need for high-throughput methods that allow quantification of site- and protein-specific 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 profiles 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.