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 (ΔF 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 ΔF 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.