Gas phase derivatization in peptide analysis I: the utility of trimethyl borate in identifying phosphorylation sites

Abstract

Phosphopeptides can readily lose H 3PO 4 during collision-activated dissociation (CAD) and this process can complicate efforts to use mass spectrometry to identify phosphorylation sites in peptides. As a part of a larger project aimed at surveying the gas phase reactivity of peptide ions, protonated phosphopeptides were derivatized in the gas phase by trimethyl borate (TMB) and subjected to CAD. During the reaction and subsequent CAD, three CH 3OH molecules are lost to give a peptide derivative that contains boron [M+B−2H] +. This ion undergoes backbone cleavages when subjected to CAD. In simple phosphopeptides, AGGsG, GsGGV, AKsF, and AKtF (lower case indicates phosphorylated residue), the boron derivatization enhances the yields of phosphate-containing sequence ions. In accord with previous work, it appears that the boron can bind to the phosphate as well as nearby nucleophilic sites and inhibit phosphate cleavage. The method was also applied to hexapeptides containing two potential phosphorylation sites (i.e., two serines), VSGAsA and LsGASA. In the former, boron derivatization leads to phosphate-containing sequence ions with greater relative intensities and provides ample information for identifying the phosphorylation site. The effect is less dramatic with LsGASA presumably because there is a greater amount of non-selective derivatization (i.e., away from the phosphate). Overall, the results suggest that boron derivatization is a potentially promising new tool for identifying phosphorylation sites in peptides where more than one site is available.