Figure 1. RP-HPLC of peptide 623 (YESEYLSK).
(A) The unmodified peptide. (B) iTRAQ-treated peptide analyzed by RP-HPLC as described in Experimental Procedures. Each of the major peaks observed at times 41.1, 45.4, and 46.5 were collected and analyzed by MALDI. The peak at 41.4 consisted of primarily peptide 623 with 1 iTRAQ label (m/z 1161.5) with a minor intensity peak representing two iTRAQ labels (m/z 1305.6), and the peak at 45.4 contained 1, 2, and to a lesser degree, 3 iTRAQ tags (m/z 1449.7) on the peptide (data not shown). The peptide fraction eluting at 46.5 min was further analyzed for iTRAQ modification(s) by MS/MS (Fig. 4B).