Fig. 5.

Cluster analysis of single peptide regulation factors revealed InlB₃₂₁-induced alterations in protein kinases. A, distribution of peptide regulation factors calculated from raw signal intensities (117 Da, InlB321; 115 Da, control) from all MS/MS scans from one Met activation experiment is plotted. The majority of the analyzed 1833 MS/MS scans are not regulated. Phosphopeptide regulations show a non-normal and shifted distribution, indicating their pronounced up-regulation in comparison with non-modified peptides. B, exemplified view of relative peptide regulations of the 15 most robustly identified protein kinases from one typical experiment. All MS/MS scans from A were normalized and iTRAQ by-product-corrected, and RFs from scans corresponding to a unique peptide were accumulated by iTRAQassist. Resulting RFs indicate Met pathway-dependent regulations of specific peptides in this small set of protein kinases after 4 min of InlB₃₂₁ treatment. The two marked peptides from MK01 (Erk2) correspond to the same amino acid sequence in its unmodified and phosphorylated form with oppositely directed RFs, whereas the two highlighted peptides from GSK3B have different sequences. The marked phosphopeptide from Nek9 comprising Thr³³³ and the doubly phosphorylated peptide from MK01 correspond to the raw data shown in Fig. 4.