Mass Spectrometric Sequencing of Proteins from Silver-Stained Polyacrylamide Gels

TLDR

Proteins from silver‑stained gels can be enzymatically digested and the resulting peptides analyzed and sequenced by mass spectrometry. A silver‑stained one‑dimensional gel of a yeast protein fraction was analyzed by nanoelectrospray tandem mass spectrometry. The method yields identical peptide maps for standard proteins from Coomassie‑ and silver‑stained gels, achieves low‑nanogram sensitivity, covers over 1000 amino acids without silver‑induced modifications, shortens sample preparation, and removes a major obstacle to low‑level sequence analysis of proteins on polyacrylamide gels.

Abstract

Proteins from silver-stained gels can be digested enzymatically and the resulting peptides analyzed and sequenced by mass spectrometry. Standard proteins yield the same peptide maps when extracted from Coomassie- and silver-stained gels, as judged by electrospray and MALDI mass spectrometry. The low nanogram range can be reached by the protocols described here, and the method is robust. A silver-stained one-dimensional gel of a fraction from yeast proteins was analyzed by nanoelectrospray tandem mass spectrometry. In the sequencing, more than 1000 amino acids were covered, resulting in no evidence of chemical modifications due to the silver staining procedure. Silver staining allows a substantial shortening of sample preparation time and may, therefore, be preferable over Coomassie staining. This work removes a major obstacle to the low-level sequence analysis of proteins separated on polyacrylamide gels.