Mass Spectrometry-Based Method for Detection and Identification of Free Thiol Groups in Proteins

Methods Mol Biol. 2019:1934:179-189. doi: 10.1007/978-1-4939-9055-9_12.

Abstract

Many proteins contain free sulfhydryl groups which can be involved in a variety of biochemical reactions. Reactive thiol groups can either reside within the active center of oxidoreductases or represent a part of a thiol-based redox switch in proteins. Therefore, the exact position of a free sulfhydryl within a protein is mostly very important.This chapter describes a mass spectrometry-based method to determine the location of protein sulfhydryl groups exemplary shown for a synthetic decapeptide and the plasma glycoprotein von Willebrand factor (VWF). We outline (1) labeling of free sulfhydryl groups, (2) enrichment of labeled peptides, and (3) detection and identification of labeled peptides by mass spectrometry.

Keywords: Cysteine; Free thiol; Mass spectrometry; Sulfhydryl group; Von Willebrand factor.

MeSH terms

  • Cysteine / chemistry
  • Disulfides / chemistry
  • Mass Spectrometry*
  • Molecular Structure
  • Peptides / chemistry
  • Proteins / chemistry*
  • Proteins / metabolism
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Sulfhydryl Compounds / chemistry*
  • Trypsin / chemistry
  • von Willebrand Factor / chemistry
  • von Willebrand Factor / metabolism

Substances

  • Disulfides
  • Peptides
  • Proteins
  • Sulfhydryl Compounds
  • von Willebrand Factor
  • Trypsin
  • Cysteine