Negative ion fragmentations of deprotonated peptides containing post‐translational modifications. An unusual cyclisation/rearrangement involving phosphotyrosine; a joint experimental and theoretical study

The characteristic fragmentations of a pTyr group in the negative ion electrospray mass spectrum of the [M–H] − anion of a peptide or protein involve the formation of PO   3 −( m/z 79) and the corresponding [(M‐H) − –HPO 3 ] − species. In some tetrapeptides where pTyr is the third residue, these characteristic anion fragmentations are accompanied by ions corresponding to H 2 PO   4 −and [(M‐H) − –H 3 PO 4 ] − (these are fragmentations normally indicating the presence of pSer or pThr). These product ions are formed by rearrangement processes which involve initial nucleophilic attack of a C‐terminal ‐CO   2 −[or ‐C(NH)O − ] group at the phosphorus of the Tyr side chain [an S N 2(P) reaction]. The rearrangement reactions have been studied by ab initio calculations at the HF/6‐31+G(d)//AM1 level of theory. The study suggests the possibility of two processes following the initial S N 2(P) reaction. In the rearrangement (involving a C‐terminal carboxylate anion) with the lower energy reaction profile, the formation of the H 2 PO   4 −and [(M‐H) − –H 3 PO 4 ] − anions is endothermic by 180 and 318 kJ mol −1 , respectively, with a maximum barrier (to a transition state) of 229 kJ mol −1 . The energy required to form H 2 PO   4 −by this rearrangement process is (i) more than that necessary to effect the characteristic formation of PO   3 −from pTyr, but (ii) comparable with that required to effect the characteristic α , β and γ backbone cleavages of peptide negative ions. Copyright © 2009 John Wiley & Sons, Ltd.