FIGURE 1.

Identification of the Ser⁴⁷⁸ phosphorylation site of CYP3A4 by MALDI-MSⁿ analyses. A, MALDI-MS spectrum of Lys-C digested CYP3A4. Some of the enzymatic peptides are labeled in the spectrum. B, MALDI-MS spectrum of Lys-C digested CYP3A4 after the phosphopeptide enrichment performed as described in the text. All the detected peaks were subjected to MS/MS analysis to identify the peptides. The majority of the observed peaks was assigned to Lys-C enzymatic peptides of CYP3A4, except the two most abundant peaks, which were observed at m/z 1770.025 and 1672.050. C, MS/MS spectrum of the peptide at m/z 1770.025. The assignment of the observed fragments revealed the identity of the peptide (477–492) from CYP3A4 with two modifications. One modification is phosphorylation of Ser⁴⁷⁸ residue. Another is modification of Lys⁴⁹² by 1 Da (see possible explanations in the text). The spectrum also revealed a prominent peak of loss of 98 Da from the parent peptide, which corresponds to the fragment with m/z 1672.050 also observed in the previous spectrum (B).