Protocol

Silver Staining, Digestion, and Extraction of Peptides from an Acrylamide Gel for MS Analysis

This protocol was adapted from "Identification of Novel Protein Complexes and Protein-Protein Interactions by Mass Spectrometry," Chapter 18, in Protein-Protein Interactions, 2nd ed. (eds. Golemis and Adams). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.

INTRODUCTION

Individual components of protein complexes separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) are visualized by silver (or Coomassie) staining. For subsequent analysis by mass spectrometry (MS), it is important to use silver staining protocols with no glutaraldehyde, as this reagent chemically modifies the protein and inhibits subsequent steps. The individual stained protein bands are excised from the gel, the proteins are enzymatically digested into peptides, and the peptides are extracted. The peptides from each protein can be subsequently analyzed by high-pressure liquid chromatography (HPLC) fractionation and electrospray ionization (ESI) MS. This generates a mass fingerprint of the peptides that comprise the unknown protein. Further structural data for peptides can be obtained by fragmenting the ionized peptides to generate a tandem mass spectrum. Alternatively, a mass fingerprint of the peptides is obtained by matrix-assisted laser desorption/ionization in combination with a time-of-flight instrument (MALDI/TOF).