Ion/ion Charge Inversion/Attachment in Conjunction with Dipolar DC Collisional Activation as a Selective Screen for Sulfo- and Phosphopeptides.

We describe a gas-phase approach for the rapid screening of polypeptide anions for phosphorylation or sulfonation based on binding strengths to guanidinium-containing reagent ions. The approach relies on the generation of a complex via reaction of mixtures of deprotonated polypeptide anions with dicationic guanidinium-containing reagent ions and subsequent dipolar DC collisional activation of the complexes. The relative strengths of the electrostatic interactions of guanidinium with deprotonated acidic sites follows the order carboxylate<phosph(on)ate<sulf(on)ate. The differences between the binding strengths at these sites allows for the use of an appropriately selected dipolar DC amplitude to lead to significantly different dissociation rates for complexes derived from unmodified peptides versus phosphorylated and sulfated peptides. The difference in binding strengths between guanidinium and phosph(on)ate versus guanidinium and sulf(on)ate is sufficiently great to allow for the dissociation of a large fraction of phosphopeptide complexes with the dissociation of a much smaller fraction of sulfopeptide complexes. DFT calculations and experimental data with model peptides and with a mixture of tryptic peptides spiked with phosphopeptides are presented to illustrate and support this approach. Dissociation rate data are presented that demonstrate the differences in binding strengths for different anion charge-bearing sites and that reveal the DDC conditions most likely to provide the greatest discrimination between unmodified peptides, phosphopeptides, and sulfopeptides.