The identification and characterization of a priori unknown proteins from an Escherichia coli (E. coli) soluble protein lysate using ion trap collision-induced dissociation of intact protein ions followed by ion/ion reactions in a quadrupole/time-of-flight tandem mass spectrometer is illustrated. The procedure involved the submission of uninterpreted product ion spectra to a peak-picking program and then to ProSightPTM for searching against an E. coli database. Examples are provided for the identification and characterization of both modified and unmodified unknown proteins with masses up to approximately 28 kDa. The availability of protein intact mass along with sequence information makes possible the characterization of proteins with post-translational modifications, such as disulfide linkages, as well as protein isoforms whose sequences are absent from a database, provided that a related form of the gene product is present in the database. This work demonstrates that the quadrupole/time-of-flight platform, in conjunction with ion-ion proton transfer reactions, can be adapted to obtain primary structure information from entire protein ions, rather than simply N- or C-terminal information from low mass-to-charge products, for proteins as large as several tens of kilodaltons.
Amino Acid Sequence Cell Extracts