title: 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 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.
authors.affiliation: Department of Translational Molecular Pathology, UT MD Anderson Cancer Center, 6767 Bertner Avenue, Houston, TX, 77030, USA. lrruhaak@gmail.com.|Department of Chemistry, UC Davis, One Shields Avenue, Davis, CA, 95616, USA. lrruhaak@gmail.com.
authors.forename: L Renee
authors.initials: LR
authors.lastname: Ruhaak
mesh_terms: D001798:Blood Proteins / Q000032:analysis / Q000737:chemistry*; D006020:Glycopeptides / Q000097:blood / Q000737:chemistry*; D006023:Glycoproteins / Q000097:blood / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D007132:Immunoglobulin Isotypes / Q000097:blood / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*
chemical_list: D001798:Blood Proteins; D006020:Glycopeptides; D006023:Glycoproteins; D007132:Immunoglobulin Isotypes
keywords: Multiple reaction monitoring; N-glycopeptides; Quantitation; RP-LC-MS; Serum