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LC-MS Analysis of (Glyco-)Proteins of Pichia pastoris

  • Clemens Grünwald-Gruber
  • Friedrich AltmannEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1923)

Abstract

In this chapter, we will present two methods for comprehensive glycoprotein characterization that are particularly but not exclusively useful for Pichia pastoris glycoproteins. One approach is intact protein mass measurement, where deglycosylation may be used to determine the mass of the unmodified protein. The other method is the classical bottom-up approach, where peptides and glycopeptides are analyzed by reversed-phase chromatography and detected by electrospray ionization mass spectrometry. The choice of chromatography solvents with a high ionic strength simplifies the identification of peaks of a particular peptide’s glycopattern as it leads to co-elution of neutral and charged, i.e., phosphorylated, glycoforms.

Key words

Ammonium formate buffer Mass spectrometry Electrospray ionization (ESI) Reversed-phase chromatography Monolithic protein column Phosphorylation 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Austrian Centre of Industrial Biotechnology (acib)ViennaAustria
  2. 2.Department of ChemistryUniversity of Natural Resources and Life Sciences Vienna (BOKU)ViennaAustria

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