Internal Sequence Analysis of Proteins Separated by Polyacrylamide Gel Electrophoresis - Hess, D.; Aebersold, R. (Elsevier)
In this report we describe a procedure for the isolation of peptide fragments derived by proteolytic cleavage of small amounts of proteins separated by polyacrylamide gel electrophoresis. Proteins resolved by gel electrophoresis were isolated by electroblotting onto a carrier matrix and digested on the support. The released peptides were separated by reverse-phase high-performance liquid chromatography and collected. Chromatography fractions were analyzed with respect to peptide homogeneity by capillary electrophoresis or electrospray mass spectrometry and, if necessary, further separated by rechromatography. Selected examples illustrate that individual steps of the procedure are mutually compatible and unnecessary sample manipulation can therefore be avoided. The described procedure is sensitive, simple, and generally applicable and is ideally suited to use in the preparation of peptide samples for internal sequencing. Peptides isolated by these protocols are equally suitable for solving other analytical problems in protein biochemistry such as the determination and localization of modified amino acid residues and the determination of catalytic residues in active sites of enzymes. Finally, we demonstrate that a detailed analysis of peptide maps can complement, and in some cases replace, protein sequencing.
http://dx.doi.org/10.1006/meth.1994.1025
Keywords: W; WW-YZ