Ingeldigestionofproteinsforpeptidefingerprintmapping

Polyacrylamide gel electrophoresis is a widely used technique to separate proteins from biological samples. Moreover, the development of two-dimensional (2D) gel electrophoresis has provided a tool for differential protein display, which allows for the quantitative analysis of many (~500-1000) of proteins simultaneously. Complex biological questions can now be approached by analyzing differences in the 2D gel patterns between control and experimental states.

While high sensitivity staining methods could visualize proteins at increasingly low levels, originally protein identification was limited by the sensitivity of Edman sequencing. However, in the last few years mass spectrometry has become established as a viable and more sensitive alternative, with the development of ESI-MS and MALDI-MS. Peptide mass fingerprinting uses the peptide masses obtained by digestion to search the protein and DNA databases, to find proteins that show a similar theoretical digest pattern (see eg., Shevchenko et al. 1996a and b).

In the protocol below, we describe the in-gel digestion procedure, as it is routinely performed in our laboratory, for peptide mass mapping of picomole to subpicomole quantities of protein derived from coomassie- or silver stained polyacrylamide gels. First, SDS is removed from the gel prior to digestion, in several ammoniumcarbonate/acetonitrile washing steps. The excised gel pieces are subsequently dried and rehydrated with enzyme in buffer. Alternatively, the washed gel pieces may be reduced and S-alkylated prior to rehydration with enzyme. After digestion, the peptides are extracted from the gel and mass analyzed.

In-gel digestion procedure