Mass spectrometric techniques for the identification tion of peptides isolated from within the polypeptide of proteins either by amino acid sequencing or by corchain after proteolysis (internal sequencing). Numerrelation of mass spectral data with sequence databases ous protocols for the generation and isolation of pepare becoming increasingly sensitive and are rapidly tides for internal sequencing from gel-separated proapproaching the limit of detection achieved by the teins have been developed (reviewed in 1). They can be staining of proteins in gels or, after electroblotting, grouped into two categories: (i) fragmentation of proon membranes. Here we present a technique for the teins on membrane supports after electroblotting and sensitive staining of proteins electroblotted onto ni-(ii) fragmentation of proteins in the gel. While there is trocellulose or polyvinylidene difluoride membranes no consensus whether on-membrane or in-gel digests and enzymatic cleavage conditions for such proteins yield higher peptide recoveries, it is generally accepted to achieve optimal recovery of peptides. The technique that samples generated by proteolysis on nitrocellulose is based on the deposition of colloidal silver on the (NC) 2 or polyvinylidenedifluoride (PVDF) membranes membrane-bound proteins. Peptide mixtures genercontain lower levels of chemical contaminants. Furated by proteolysis on the membrane were recovered thermore, on-membrane cleavage protocols are faster, at high yields and were compatible with analysis by easier to automate, and yield smaller sample volumes.
reverse-phase chromatography and on-line electro-
The rapid progress in protein and peptide mass specspray ionization mass spectrometry. This simple and trometry, in particular the development of the electrorapid colloidal silver staining procedure allowed the spray (ESI) and matrix-assisted laser desorption visualization of less than 5 ng of protein in a band and (MALDI) ionization techniques, has led to the developthus approached the sensitivity of silver staining in ment of alternative techniques for protein identificagels. We demonstrate that this method allows the detection of subpicomole amounts of electroblotted pro-tion. In addition to chemical stepwise degradation, teins and their identification by high-performance liq-amino acid sequences of peptides can be determined by uid chromatography-electrospray ionization tandem tandem mass spectrometry (MS/MS), either by partial mass spectrometry. ᭧ 1997 Academic Press interpretation of the tandem mass spectra or by sequence correlation with the rapidly growing sequence databases. Several computer algorithms have been developed and successfully applied that permit conclu-The determination of the primary structure of prosive protein identification by the collective of a number teins separated by one-or two-dimensional polyacrylamide gel electrophoresis is an essential step in many 2 Abbreviations used: NC, nitrocellulose; PVDF, polyvinylidenedprojects in protein biochemistry and cell biology. Tradiifluoride; MS/MS: tandem mass spectrometry; ESI, electrospray ion- tionally, sequences were determined by the stepwise ization; MALDI, matrix-assisted laser desorption ionization; CID, collision-induced dissociation; AB, ammonium bicarbonate; NEM, N-chemical degradation of proteins from the N-terminal ethylmorpholine; CA, carbonic anhydrase; BSA, bovine serum albumin; RP-HPLC: reverse-phase-high-performance liquid chromatography; m/z, mass-to-charge ratio; CE, capillary electrophoresis;