Phosphate Migration versus the Loss of Phosphoric Acid in Protonated Phosphopeptides: A Computational Study.

Residue-specific phosphorylation is a protein post-translational modification that regulates cellular functions. Experimental determination of the exact sites of protein phosphorylation provides an understanding of the signaling and processes at work for a given cellular state. Any experimental artifact that involves migration of the phosphate group during measurement is a concern, as the outcome can lead to erroneous conclusions that may confound studies on cellular signal transduction. Herein, we examine computationally the mechanism by which a phosphate group migrates from one serine residue to another serine in monoprotonated pentapeptides [B A -pSer-Gly-Ser-B B + H] + → [B A -Ser-Gly-pSer-B B + H] + (where B A and B B are different combinations of the three basic amino acids, histidine, lysine, and arginine). In addition to moving the phosphate group, the overall mechanism involves transferring a proton from the N-terminal amino acid, B A , to the C-terminal amino acid, B B . This is not a synchronous process, and there is a key high-energy intermediate, structure C , that is zwitterionic with both the basic amino acids protonated and the phosphate group attached to both serine residues and carrying a negative charge. The barriers to moving the phosphate group are calculated to be in the range of 219-274 kJ mol -1 at the B3LYP/6-31G(d) level. These barriers are systematically slightly lower and in good agreement with single-point energy calculations at both M06-2 X /6-311++G(d,p) and MP2/6-31++G(d,p) levels. The competitive reaction, loss of phosphoric acid from the protonated pentapeptides, has a barrier in the range of 176-202 kJ mol -1 at the B3LYP/6-31G(d) level. Extension of the theory to M06-2 X /6-311++G(d,p)//B3LYP/6-31G(d) and MP2/6-31++G(d,p)// B3LYP/6-31G(d) gives higher values for the loss of phosphoric acid, falling in the range of 196-226 kJ mol -1 ; these are comparable to the barriers against phosphate migration at the same levels of theory. For larger peptides His-pSer-(Gly) n -Ser-His, where n has values from 2 to 5, the barriers against the loss of phosphoric acid are higher than those against the phosphate group migration. This difference is most pronounced and significant when n = 4 and 5 (the differences are approximately 80 kJ mol -1 under the single-point energy calculations at the M06-2X and MP2 levels). Energy differences using two more recent functionals, M08-HX and MN15, on His-pSer-(Gly) n -Ser-His, where n = 1 and 5, are in good agreement with the M06-2X and MP2 calculations. These results provide the mechanistic rationale for phosphate migration versus other competing reactions in the gas phase under tandem mass spectrometry conditions.