Table 2.
Tandem mass spectrum of LysC phosphopeptide 630–644 from phosphorylated recombinant myosin
| Residue | a and b ions |
y ions |
||||
| Ion | Calculated | Observed | Ion | Calculated | Observed | |
| Ala630 | a1 | 44.050 | 44.049 | — | — | — |
| Ala631 | a2 | 115.087 | 115.090 | y14 | 1,454.697 | ND |
| b2 | 143.082 | 143.085 | ND | |||
| Ala632 | b3 | 214.119 | 214.116 | y13 | 1,383.660 | ND |
| Gly633 | b4 | 271.141 | 271.143 | y12 | 1,312.623 | ND |
| Gly634 | b5 | 328.162 | 328.154 | y11 | 1,255.602 | ND |
| Ser635 | b6 | 415.194 | ND | y10 | 1,198.580 | ND |
| Arg636 | b7 | 571.295 | 571.301 | y9 | 1,111.548 | ND |
| Asn647 | b8 | 685.338 | 685.359 | y8 – NH3 - H3PO4 | 840.433 | 840.458 |
| Arg638 | b9 | 841.439 | 841.439 | y7 - H3PO4 | 743.417 | 743.416 |
| pSer639 | b10 - H3PO4 | 910.461 | 910.459 | y6 - H3PO4 | 587.316 | 587.326 |
| b10 | 1,008.438 | ND | y6 | 685.303 | ND | |
| Thr640 | b11 | 1,109.485 | ND | y5 | 518.305 | 518.309 |
| Gly641 | b12 | 1,166.507 | ND | y4 | 417.257 | 417.270 |
| Arg642 | b13 | 1,322.608 | ND | y3 | 360.235 | 360.248 |
| Gly643 | b14 | 1,379.629 | ND | y2 | 204.134 | 204.141 |
| Lys644 | — | — | — | y1 | 147.113 | 147.112 |
The measured mass of the peptide was 1,524.72, and the calculated mass for the monophosphorylated peptide is 1,524.73. The doubly charged ion with m/z 763.371 was sequenced. The dominant product ion in the spectrum was at 714.382 and was produced by neutral loss of H3PO4 from the peptide. However, there was sufficient abundance of other peaks to identify Ser639 unambiguously as the phosphorylated residue. Fragmentation of the peptide generated ions whose mass allowed sequencing in both the amino-to-carboxyl direction (a or b ions) and the carboxyl-to-amino direction (y ions). The observed mass of the y5 ion established that Thr640 is not phosphorylated, whereas the masses of the y6 ions with neutral loss of phosphate established that Ser639 was phosphorylated. ND, the ion was not detected.