High-Throughput Analysis of IgG Fc Glycopeptides by LC-MS
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 wordsLiquid 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
- 10.Bruker Daltonics. https://www.bruker.com/products/mass-spectrometry-and-separations/ms-software/compass/overview.html. Accessed 11 Mar 2015
- 13.Van Rossum G, Drake FL Jr (1995) Python reference manual. Centrum voor Wiskunde en Informatica, AmsterdamGoogle Scholar
- 17.Python(x,y). http://python-xy.github.io/. Accessed 21 Aug 2015
- 18.Anaconda. https://www.continuum.io/downloads. Accessed 14 July 2015
- 24.Pluskal T, Castillo S, Villar-Briones A, et al. (2010) MZmine 2: modular framework for processing, visualizing, and analyzing mass spectrometry-based molecular profile data. BMC Biochem 11:395Google Scholar
- 26.Geany. http://www.geany.org/. Accessed 15 Oct 2015
- 27.IBM SPSS Statistics. http://www-01.ibm.com/software/analytics/spss/products/statistics/. Accessed 3 Nov 2015
- 28.Microsoft Excel. https://products.office.com/en-us/excel. Accessed 3 Nov 2015