Absolute and site-specific quantification of protein phosphorylation using integrated elemental and molecular mass...

The validity of using elemental phosphorus standards to accurately and precisely quantify phosphopeptides by capillary HPLC (capHPLC) coupled to ICP-collison cell-MS is investigated in detail. Operating requirements to maintain stable (31)P sensitivity along the reversed-phase gradient are described. Specifically, the use of an optimum postcolumn makeup flow with a defined acetonitrile content turned out to be necessary to buffer the acetonitrile variation of the capillary chromatographic eluent and ensure plasma stability. Then, a highly pure P-containing standard (bis(4-nitro-phenyl) phosphate, BNPP) was spiked into the samples and used to quantify them with very low absolute errors (2-4%) and excellent precision (3-6%). The capHPLC-ICPMS method showed excellent linearity over 3 orders of magnitude and provided adequate detection limits (110 fmol, 3.4 pg P). Accurate quantification of the phosphopeptides present in a tryptic digest of beta-casein and casein from bovine milk was then attempted. Previously, and in order to be able to close mass balances, total P contents, percentages of inorganic P present, and recoveries from the reversed-phase column used in the separation were computed for each sample. Quantification using the spiked BNPP for the different phosphopeptides detected matched the expected values well validating the quantitative methodology proposed. The capHPLC-ESIMS analysis allowed elucidating amino acid sequences, a requisite still necessary to translate the determined amount of P in each chromatographic peak into amount of phosphopeptide. The great potential of these strategies, based on ICPMS detection, to assess the many procedures proposed and commonly used for purification, preconcentration, and/or isolation of phosphopeptides in phosphoproteomics studies is demonstrated using a commercially available titanium dioxide (TiO(2)) cartridge for phosphopeptide enrichment from complex mixtures. Quantitative results obtained allow one to assess individual phosphopeptide recoveries from the TiO(2) cartridge with unsurpassed accuracy. Of course, this information is essential for reliable absolute quantifications in phosphoproteomics.