Protocol

In-Gel S-Pyridylethylation of Gel-Resolved Proteins: Whole Gel Method

This protocol was adapted from "Peptide Mapping and Sequence Analysis of Gel-Resolved Proteins," Chapter 7, in Proteins and Proteomics by Richard J. Simpson. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.

INTRODUCTION

This protocol describes a method for performing reduction and S-alkylation of Coomassie-blue-stained proteins within an intact gel. Reduction is performed with dithiothreitol, and alkylation with 4-vinylpyridine. (Treatment of free cysteines with 4-vinylpyridine yields the S-β-(4-pyridylethyl) cysteinyl derivative.) S-β-(4-pyridylethyl) cysteine-containing peptides can be readily identified during RP-HPLC by their characteristic absorbance at 254 nm and during electrospray ionization tandem mass spectrometry by the appearance of a characteristic pyridylethyl fragment ion of 10⁶ Da. The position of cysteine residues in a polypeptide sequence can be determined either by mass spectrometry or as phenylthiohydantoin S-β-(4-pyridylethyl) cysteine during Edman degradation.