Mass Spectrometric Sequencing of Proteins from Silver-Stained Polyacrylamide Gels

Abstract

Proteins from silver-stained gels can be digested enzymatically and the resulting peptide 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 nano-electrospray 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.

Proteomic analysis of human vitreous fluid by fluorescence-based difference gel electrophoresis (DIGE): a new strategy for identifying potential candidates in the pathogenesis of proliferative diabetic retinopathy

“…A pool consisting of equal amounts of each of the samples analysed in the experiment was prepared as an internal standard for quantitative comparisons [14]. To avoid any possible bias introduced by labelling efficiency, half of the samples from each group were labelled with Cy3 dye and the other half with Cy5 dye.…”

“…In-gel trypsin digestion was performed as described previously [14], using autolysis-stabilised trypsin (Promega, Madison, WI, USA). Tryptic digests were purified using ZipTip microtitre plates (Millipore, Billerica, MA, USA).…”

Changes in disulfide bond content of proteins in a yeast strain lacking major sources of NADPH

“…Proteins of interest that corresponded to proteins labeled with 6-IAF or to carbonylated proteins were excised robotically using a Proteome Works spot cutter (Bio-Rad) and digested in situ with trypsin according to standard protocols based on the initial work of Mann and co-workers [23]. Briefly, protein spots were excised from the gel and destained in 50% acetonitrile/40 mM ammonium bicarbonate, pH 7.4.…”

Sperm-associated antigen 1 is expressed early in pancreatic tumorigenesis and promotes motility of cancer cells

“…Protein bands at approximately 104 kDa were visualized by silver staining and excised from 1D SDS-polyacrylamide gel electrophoresis (PAGE) 12% Gels (18 18 cm  1 mm) that were run on a BioRad Protean II System, as described previously (Shevchenko et al, 1996). The bands were subsequently trypsin digested and analysed using mass spectrometry (MS) as described in Cutillas et al (2004).…”