ABSTRACT
A method for the characterization of proteins separated by isoelectric focusing in carrier ampholytes (CA-IEF) or immobilized pH gradient (IPG) gels by in-gel digestion and mass spectrometry is described. Proteins are detected by an improved imidazole-Sodium dodecyl sulfate (SDS)-zinc staining adapted for IEF and IPG gels. Sensitivity is close to that of mass spectrometry-compatible silver staining, but simpler and faster. Proteins were digested in imidazole-SDS-zinc stained CA-IEF and IPG gels in the presence of a zinc-chelating agent. Mass spectra were clearly interpretable as carrier ampholytes which were efficiently removed before digestion; high-sequence coverage that allowed isoform characterization was obtained by analyzing both the aqueous and the organic phase extracts.
Subject(s)
Proteins/analysis , Ampholyte Mixtures , Animals , Humans , Hydrogen-Ion Concentration , Imidazoles , Isoelectric Focusing/methods , Protein Isoforms/analysis , ZincABSTRACT
Identification and characterization of proteins isolated from natural sources by polyacrylamide gel electrophoresis has become a routine technique. However, efficient sample proteolysis and subsequent peptide extraction is still problematic. Here, we present an improved protocol for the rapid detection of polyacrylamide gel-separated proteins, in situ protein modification, proteolytic digestion and peptide extraction for subsequent protein identification and characterization by capillary high-performance liquid chromatography/tandem mass spectrometry. This simple technique employs the rapid imidazole-zinc reverse stain, in-gel S-pyridylethylation and proteolytic digestion of microcrushed polyacrylamide gel pieces with proteases. This technique obviates the need for buffer exchange or gel lyophilisation due to all of the sample manipulation steps being carried out at near neutral pH and thus lends itself readily to automation.
