Observations on the quantitation of the phosphate content of peptides by fast-atom bombardment mass spectrometry.

Equimolar mixtures of the phosphorylated and dephosphorylated forms of several peptides have been subjected to fast-atom bombardment mass spectrometry (FABMS), to investigate whether the stoichiometry of phosphorylation can be determined from the relative molecular-ion abundances of the phospho and dephospho derivatives. It is concluded that quantitation can be achieved for peptides with large positive or negative hydrophobicity/hydrophilicity indices (delta F values) where addition of a phosphate group does not alter the distribution of the peptide within the matrix significantly. For peptides with small positive or negative delta F values, phosphopeptides tend to be partially suppressed by their dephosphorylated counterparts. Suppression can be partially or totally overcome by conversion of the peptide to a hydrophobic derivative, and by the selection of an appropriate matrix. Alternatively, addition of a very strong acid, perchloric acid, can even reverse the original suppression effect. This last effect is believed to be due to the increased ionic strength in the matrix, which forces a relatively hydrophilic analyte to the matrix surface; and the ability of such a phosphorylated analyte to form a more stable gas-phase cation.