The poor resolution of spots 1, 2 and 3 caused some problems. Spot 2 (PduG) was located below spot 1 (PduC) which seemed incorrect since PduG is has an apparent molecular mass 4 kDa greater than that of PduC. In addition the N-terminal sequence of spot 3 did not match any proteins encoded by the S. enterica genome. To resolve these problems the polyhedral organelles were separated on a larger gel and the resulting protein spots were then subjected to MALDI-TOF analyses (see below).


Table 4-3. Organelle proteins identified using N-terminal sequencing
     Spot         Experimental N-     Predicted N-        Corresponding
                  terminal sequence   terminal sequence* Protein
1                 MRSKRFE             MRSKRFE            PduC
2                 MRYIAGL             MRYIAGL            PduG
3                 AEKELVL             ND                  ?
4                 SSNELVE             MSSNELV            PduB
5                 AEKSCSL             MAEKSCS            PduB'
6                 MNTDAIE             MNTDAIE            PduE
7                 MQQEALG             MQQEALG            PduA
8                 MNLALGL             MNNALGL            PduJ
* Prediction based on DNA sequence. ND not determined. MALDI-TOF Analysis

     Proteins corresponding to spots 1-9, 11, 13-15 and 17-20, (Figure 4-5) were excised, digested with trypsin, and analyzed by MALDI-TOF MS and protein mass fingerprinting (Table 4-4). A sample spectrum of spot 11 is shown in Figure 4-6. Both the MS-Fit and Profound Web-based programs were used to analyze the MALDI-TOF spectra. The result of a database search was considered significant if it was ranked as best hit and there was a sequence coverage of at least 25% in either program. Additionally, a MOWSE score (MS-Fit) of at least le+003 or a Z-score (Profound) greater than or equal to 1.65 was needed (Baker and Clauser, Zhang and Chait 2000). Based on these analyses, the observed protein spots were identified as the following S.