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High-Throughput Analysis of IgG Fc Glycopeptides by LC-MS

  • David Falck
  • Bas C. Jansen
  • Noortje de Haan
  • Manfred WuhrerEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1503)

Abstract

This chapter contains a nanoscale liquid chromatography–mass spectrometry method for the glycoform profiling of the conserved Fc N-glycosylation site of monoclonal and polyclonal immunoglobulin G (IgG). It describes in detail LaCyTools, a program for automated data (pre-)processing of the obtained LC-MS data. The minimal sample preparation necessary is explained as well as an optional method for affinity purification of (polyclonal) antibodies from serum or plasma.

After (optional) affinity purification, the pure IgG is cleaved with trypsin. The tryptic glycopeptides are separated almost exclusively on their peptide backbone. This ensures similar response factors for all glycoforms in the MS detection and allows the collection of separate glycoform profiles for different IgG isoforms or allotypes. LaCyTools automatically performs label-free (relative) quantitation of the obtained data after minimal manual input and additionally calculates several quality criteria which can be used for data curation at the level of both individual analytes and entire LC-MS runs.

Key words

Liquid chromatography–mass spectrometry (LC-MS) Glycopeptide analysis Immunoglobulin G High-throughput glycosylation profiling Glycoform profiling Bottom-up proteomics Glycoproteomics Automated data analysis Label-free quantification Quality scores 

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • David Falck
    • 1
  • Bas C. Jansen
    • 1
  • Noortje de Haan
    • 1
  • Manfred Wuhrer
    • 1
    • 2
    Email author
  1. 1.Center for Proteomics and MetabolomicsLeiden University Medical CenterLeidenThe Netherlands
  2. 2.Division of BioAnalytical ChemistryVU University AmsterdamAmsterdamThe Netherlands

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