We and other groups have long recognized the benefit of using charged hydrophobic derivatives to facilitate analysis of peptides by FAB-CAD-MS/MS. The analytical benefits of the charged derivative for structure elucidation, however, have been offset by difficulties in derivatization of low levels of the analyte prior to analysis. The goal of this project is to improve the chemistry to the extent that the charged derivative can be prepared reliably at low level of sample in a biological extract. We have found that the tris (2,4,6-trimethoxyphenyl)-phosphonium (TMPP) derivative can be prepared easily and reliably with an activated acetic acid pentafluorylthiophenyl ester. This reagent can be used in a one-step derivatization reaction to achieve better than 90% conversion of the peptide at the low picomole level. Because of the high reactivity of the reagent it is used in only 10-fold excess and thus requires minimal or no cleanup prior to direct analysis of the reaction mixture by FAB or MALDI-MS. CAD-MS/MS of the TMPP derivative yields a subset of possible bond cleavage fragments, with dominant A-type ions accompanied by D- and C-type ions.
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