Phosphopeptide anion characterization via sequential charge inversion and electron-transfer dissociation.

Sequential ion/ion reactions have been used to characterize phosphopeptides present in relatively simple peptide mixtures, including one generated from the tryptic digestion of alpha-casein. The phosphopeptides in these mixtures gave rise to either low or no signals via positive ion electrospray ionization. Strong signals, however, were generated in the negative ion mode. An initial ion/ion reaction that employed multiply protonated amino-terminated dendrimers converted phosphopeptide anions to the doubly protonated species. The doubly charged cations were then subjected to ion/ion electron transfer to induce dissociation. Electron-transfer dissociation of doubly positively charged phosphopeptides yields characteristic c- and z-type fragment ions by dissociation of the N-C(alpha) bond along the peptide backbone while preserving the labile posttranslational modifications. These results illustrate the ability to alter ion charge after ion formation and prior to structural interrogation. Phosphopeptides provide an example where it can be difficult to form strong doubly charged cation signals directly when they are present in mixtures, which, as a result, precludes the use of electron-transfer dissociation as a structural probe. The sequential ion/ion reaction process described here, therefore, can provide a new capability for structural interrogation in phosphoproteomics.