Due to low abundance, some spots could not be identified unambiguously, revealing a drawback of working with gel-based proteomics. Phase 2 flagellin was downregulated in the luxS mutant, corresponding to what was previously reported by Karavolos et al. . An intriguing observation was the fact that two distinct protein spots, NVP-BSK805 chemical structure absent MEK inhibitor cancer in the luxS mutant as compared to wildtype, were identified by mass spectrometry as being LuxS. This
result led us to investigate the LuxS protein itself in more detail. Figure 1 Image of the master gel used in the 2D-DIGE analysis comparing the proteome of wildtype S. Typhimurium with that of a luxS mutant. Spots with white spot boundaries were differentially expressed. The numbers indicated, correspond to the spot numbers in Table 1. Table 1 Differentially expressed spots in the 2D-DIGE analysis Spot nr.a Name Description Protein IDb Av.
Ratioc p-valued luxS mutant vs. wildtype 1 LuxS S-ribosylhomocysteine lyase Q9L4T0 -13.50 9.80E-04 2 LuxS S-ribosylhomocysteine lyase Q9L4T0 -9.77 1.70E-03 3 n.i. n.i n.i. -3.94 7.00E-03 4 FljB Phase 2 flagellin P52616 -2.11 5.00E-04 5 FljB Phase 2 flagellin P52616 -1.75 8.00E-04 6 n.i. n.i. n.i. -1.72 1.40E-03 a Corresponding spot number on the gel image in Figure 1 b Protein identification number c Average fold increase (positive ratio) or decrease (negative ratio) in expression of a protein in the mutant compared to the wildtype d P-value of the t-test analysis comparing the mutants to the wildtype n.i. indicates not identified LuxS modification Fenbendazole Based on the relative position of the two LuxS spots on the gels and the theoretical pI of LuxS as calculated with ScanSite Vorinostat molecular weight pI/MW, the most basic (right) spot (Figure 2A) corresponds to the native LuxS form while the other spot corresponds to LuxS with an additional negative charge. Efforts to identify the nature of this modification by tandem mass spectrometry were unsuccessful. Phosphorylation
is a common posttranslational modification that induces a protein shift to the acidic side of 2D gels due to the negative charge of the phosphate group. Moreover, LuxS proteins from several Gram-negative bacteria contain a semi-conserved tyrosine phosphorylation site motif . This led us to investigate whether the modification of LuxS in S. Typhimurium corresponds to a tyrosine phosphorylation. First, we attempted to detect a phosphorylated form of LuxS using the phosphospecific ProQ-Diamond stain (Invitrogen) on a 2D gel. However, no LuxS spot could be detected in this way (data not shown). Secondly, Western blotting using anti-phosphotyrosine antibodies was performed on an immunoprecipitated LuxS protein fraction. This immunoprecipitation step increases the LuxS concentration to facilitate detection of a putative phosphorylated form. Yet, LuxS could not be detected by these antibodies, making a tyrosine phosphorylation unlikely (data not shown).