Figure 2.

Different phosphorylation states and N-terminal modifications are found in RTP isoforms. A, Enriched phosphopeptides derived from the α spot in Figure 1B were subjected to on-target alkaline phosphatase treatment using MALDI-QIT-TOF MS. The α spot contained a peptide ion at m/z 2111.8 corresponding to peptide 3–19 of RTP (top trace). Two peaks, corresponding to single and double loss of 98 Da, characteristic of the loss of one or two neutral H₃PO₄, were also present. After on-target alkaline phosphatase treatment (bottom trace), a dephosphorylated peptide was generated by the loss of a phosphate group (HPO₃; −80 Da). B, Enriched phosphopeptides derived from the β spot in Figure 1B were subjected to on-target alkaline phosphatase treatment using MALDI-QIT-TOF MS. The β spot contained two peptide ions at m/z 2102.8 and m/z 2031.7, corresponding to RTP peptides 2–19 and 3–19, respectively (top trace), each showing single loss of 98 Da caused by loss of H₃PO₄. After on-target alkaline phosphatase treatment (bottom trace), a dephosphorylated peptide was generated by the loss of a phosphate group (HPO₃; −80 Da). C, MS/MS analyses on the RTP N-terminal peptide ions m/z 1919.7, 1948.9, and 1990.8. The N-terminal amino acid in the m/z 1919.8 was acetylated Met3, the N-terminal amino acid in the m/z 1948.9 was unmodified Ala2, and N-terminal amino acid in m/z 1990.8 ion was acetylated Ala2.