Figures 33-34 illustrate the use of MALDI/TOF/MS to map the peptide sequence of a tryptic digest to identify the original protein (42). In this case, two bands of different molecular weight (MW) on SDS-PAGE were observed in genetically manipulated and native insulinoma cells that were immunoreactive with antibodies to the Group VIA Phospholipase A2 (iPLA2b) on Western blotting (Figure 33). The higher MW material corresponded to the product expected from the cDNA sequence and was overexpressed in cells stably transfected with the iPLA2b cDNA in a retroviral vector (OE). In contrast, only the lower MW material was observed in native insulinoma cells (832/13) or cells transfected with empty vector (V). The question arose as to whether the lower MW material was a product of the iPLA2b gene or simply a cross-reactive protein encoded by another gene.
To address this question, the bands were excised from the gels, digested with trypsin, and analyzed by MALDI/TOF/MS (Figure 34). For both higher and lower MW bands, at least ten ions corresponded exactly in m/z value to tryptic peptides expected from the iPLA2b cDNA sequence, providing strong evidence that the lower MW band is in fact a product of the iPLA2b gene. This was confirmed by LC/ESI/MS/MS analyses of several of the peptides on a QTOF instrument (Figure 35), which produced spectra that confirmed the amino acid sequence expected from the iPLA2b cDNA sequence. Although a doubly charged ion was subjected to CAD, the QTOF data system converts the data into a spectrum for singly charged species to facilitate interpretation. Singly charged species themselves are not subjected to CAD and are excluded in the data-dependent MS to MS/MS transition algorithm because most singly charged species represent chemical noise rather than bona fide tryptic peptides. Most tryptic peptides will have a charge state of at least +2 because they will have a C-terminal Lys or Arg residue with a basic side chain and an N-terminal alpha amino group, and both of these moieties are expected to be protonated under the conditions of the LC/ESI/MS/MS analyses.