Identification of in-gel digested proteins by complementary peptide mass fingerprinting and tandem mass spectrometry data obtained on an electrosprayionization quadrupole time-of-flight mass spectrometer

The present study reports a procedure developed for the identification of S DS-polyacrylamide gel electrophoretically separated proteins using an elect rospray ionization quadrupole time-of-flight mass spectrometer (Q-TOF MS) e quipped with pressurized sample introduction. It is based on in-gel digesti on of the proteins without previous reduction/alkylation and on the capabil ity of the Q-TOF MS to provide data suitable for peptide mass fingerprintin g database searches and for tandem mass spectrometry (MS/MS) database searc hes (sequence tags), Omitting the reduction/alkylation step reduces sample contamination and sample loss, resulting in increased sensitivity. Omitting this step can leave disulfide-connected peptides in the analyte that can l ead to misleading or ambiguous results from the peptide mass fingerprinting database search. This uncertainty, however, is overcome by MS/MS analysis of the peptides, Furthermore, the two complementary MS approaches increase the accuracy of the assignment of the unknown protein. This procedure is th us, highly sensitive, accurate, and rapid. In combination with pressurized nanospray sample introduction, it is suitable for automated sample handling . Here, we apply this approach to identify protein contaminants observed du ring the purification of the yeast DNA mismatch repair protein M1h1.