Unit

UNIT 10.25 Preparation of Proteins and Peptides for Mass Spectrometry Analysis in a Bottom-Up Proteomics Workflow

  1. Rebekah L. Gundry,
  2. Melanie Y. White,
  3. Christopher I. Murray,
  4. Lesley A. Kane,
  5. Qin Fu,
  6. Brian A. Stanley,
  7. Jennifer E. Van Eyk

Published Online: 1 OCT 2009

DOI: 10.1002/0471142727.mb1025s88

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Gundry, R. L., White, M. Y., Murray, C. I., Kane, L. A., Fu, Q., Stanley, B. A. and Van Eyk, J. E. 2009. Preparation of Proteins and Peptides for Mass Spectrometry Analysis in a Bottom-Up Proteomics Workflow. Current Protocols in Molecular Biology. 88:VI:10.25:10.25.1–10.25.23.

Author Information

  1. The Johns Hopkins University School of Medicine, Baltimore, Maryland

Publication History

  1. Published Online: 1 OCT 2009
  2. Published Print: OCT 2009

Abstract

This unit outlines the steps required to prepare a sample for MS analysis following protein separation or enrichment by gel electrophoresis, liquid chromatography, and affinity capture within the context of a bottom-up proteomics workflow in which the protein is first broken up into peptides, either by chemical or enzymatic digestion, prior to MS analysis. Also included are protocols for enrichment at the peptide level, including phosphopeptide enrichment and reversed-phase chromatography for sample purification immediately prior to MS analysis. Finally, there is a discussion regarding the types of MS technologies commonly used to analyze proteomics samples, as well as important parameters that should be considered when analyzing the MS data to ensure stringent and robust protein identifications and characterization. Curr. Protoc. Mol. Biol. 88:10.25.1-10.25.23. © 2009 by John Wiley & Sons, Inc.

Keywords:

  • in-solution digestion;
  • in-gel digestion;
  • peptide desalting proteomics;
  • mass spectrometry