Figure 4. Identification of alternative processing of histone variant H2AFX.

ETD fragmentation spectra corresponding to four differentially processed and modified N-termini of the isoform H2AFX (GI: 4504253). The positions of z,y (C-terminal) and c (N-terminal) fragment ions are indicated in the spectra and in the presented peptide sequences with the lower case (ac), (ac/me3) (me1), (me2), (ph) and (ub) corresponding to modifications of the amino acids by acetylation, acetylation/trimethylation, monomethylation, dimethylation, phosphorylation and ubiquitination, respectively. (A) ETD spectrum of the ion (M+3H)³⁺ at m/z 377.7 corresponding to the peptide with the amino acid positions 2–12 in H2AFX. The initial methionine has been removed, the N-terminal serine acetylated and the arginine in the peptide was monomethylated. (B) ETD spectrum of the peptide corresponding to the amino acid positions 5–18; the ion (M+3H)³⁺ at m/z 570.2. The peptide is modified by acetylation/trimethylation, phosphorylation, ubiquitination and two dimethylations. The mass difference of 181 between the fragment ions c3 and c4 corresponds to the phosphorylated threonine. The mass difference of 242 between the fragment ions c5 and c6 corresponds to the ubiquitinated lysine. The mass difference of 156 between the fragment ions c11 and c12 corresponds to the dimethylated lysine. The mass difference of 170 between the fragment ions z12 and z13 corresponds to the acetylated/trimethylated lysine. (C) ETD spectrum of the peptide corresponding to the amino acid positions 5–18; the ion (M+3H)³⁺ at m/z 567.4. The initial glycine has been acetylated after cleavage of the four N-terminal amino acids of the protein. The peptide is also modified by a monomethylation and two ubiquitinations. The mass differences of 242 between the fragment ions z2 and z3, z12 and z13 and c11 and c12, as well as the mass difference of 121 between the doubly charged fragment ions c11²⁺ and c12²⁺ correspond to the ubiquitinated lysines. (D) ETD spectrum of the peptide corresponding to the amino acid positions 5–18; the ion (M+3H)³⁺ at m/z 527.6. The N-terminal glycine is acetylated. The peptide is also modified by monomethylation, phosphorylation and dimethylation. The mass difference of 156 between the fragment ions c9 and c10 corresponds to the dimethylated lysine. The mass difference of 167 between the fragment ions c12 and c13 corresponds to the phosphorylated serine.