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. 2012 Dec 17;110(1):E23–E32. doi: 10.1073/pnas.1219713110

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.