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

  • L. Renee Ruhaak
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1503)

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.

Key words

N-glycopeptides Quantitation Serum Multiple reaction monitoring RP-LC-MS 

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Department of Translational Molecular PathologyUT MD Anderson Cancer CenterHoustonUSA
  2. 2.Department of ChemistryUC DavisDavisUSA

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