Abstract
Publisher Summary Recently, there has been nearly a 10-fold increase in the sensitivity, at which internal sequencing can routinely carry out on “unknown” proteins. The study discussed in this chapter have evaluated an in gel digest protocol, such that critical steps in this procedure can be identified and optimized, and such that realistic limits can be placed on the amount of protein required to maintain a success rate that approaches 100%. With the exception of studies on bovine serum albumin (BSA) and human transferrin, all other digests considered in this study are carried out on Coomassie Blue-stained gel bands that had been excised from sodium dodecyl sulfate (SDS) polyacrylamide gels. The BSA and transferrin samples are subject to SDS-PAGE and are otherwise prepared as described in the chapter. Proteins are quantified by subjecting 10–15% aliquots of all gel slices to hydrolysis and ion exchange amino acid analysis. Difficulty in obtaining high sensitivity matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) spectra on in gel digests (for the purpose of peptide mass database searching prior to HPLC fractionation) carried out in the presence of Tween 20 provided the impetus for determining if this detergent is indeed essential. Based on tryptic and lysyl endopeptidase digests of transferrin (25 pmol), Tween 20 (0.02%) did not have any significant impact on overall peptide yield as judged by the resulting absorbance profiles. Based on the overall data obtained it becomes evident that gel digestion is a remarkably robust approach for obtaining internal peptide sequences from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins.
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