Identification of in-gel digested proteins by complementary peptide mass fingerprinting and tandem mass spectrometry data obtained on an electrospray ionization quadrupole time-of-flight mass spectrometer.
The present study reports a procedure developed for the identification of SDS-polyacrylamide gel electrophoretically separated proteins using an electrospray ionization quadrupole time-of-flight mass spectrometer (Q-TOF MS) equipped with pressurized sample introduction. It is based on in-gel digestion of the proteins without previous reduction/alkylation and on the capability of the Q-TOF MS to provide data suitable for peptide mass fingerprinting database searches and for tandem mass spectrometry (MS/MS) database searches (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 lead 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 thus, 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 during the purification of the yeast DNA mismatch repair protein Mlh 1.
Borchers
C
C
Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Peter
J F
JF
Hall
M C
MC
Kunkel
T A
TA
Tomer
K B
KB
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D000070957:MutL Protein Homolog 1; D010449:Peptide Mapping / Q000379:methods*; D029701:Saccharomyces cerevisiae Proteins
D048868:Adaptor Proteins, Signal Transducing; D005656:Fungal Proteins; C088859:MLH1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000070957:MutL Protein Homolog 1