A simplified procedure for the reduction and alkylation of cysteine residues in proteins prior to proteolytic digestion and mass spectral analysis

Anal Biochem. 2004 Oct 1;333(1):174-81. doi: 10.1016/j.ab.2004.04.013.


A procedure for reduction and alkylation of cysteine residues in proteins was developed using the volatile reagents triethylphosphine and iodoethanol. These reagents may be used to modify proteins in solution, as well as proteins in gel slices, prior to proteolytic digestion and mass spectral analysis. The procedure eliminates several steps with both types of samples. Samples in solution do not need to be desalted following reduction and alkylation, with excess reagent being removed under vacuum. For gel slices, the procedure combines washing, destaining, reduction and alkylation into a single step. The procedure was applied successfully to samples as complex as serum, and we demonstrated alkylation of cysteines to be quantitative in purified proteins. We also were able to reduce and alkylate proteins with these reagents during the gas phase. Elimination of the need for desalting of samples after reaction raised the possibility of automation of the procedure for liquid samples, which is difficult with conventional reduction and alkylation chemistries.

MeSH terms

  • Alkylating Agents / chemistry
  • Alkylation
  • Animals
  • Chickens
  • Cysteine / chemistry*
  • Electrophoresis, Gel, Two-Dimensional
  • Ethanol / analogs & derivatives
  • Ethanol / chemistry
  • Muramidase / chemistry*
  • Oxidation-Reduction
  • Peptide Mapping / methods
  • Phosphines / chemistry
  • Rats
  • Serum / chemistry*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods*


  • Alkylating Agents
  • Phosphines
  • Ethanol
  • Muramidase
  • Cysteine