Absolute and site-specific quantification of protein phosphorylation using integrated elemental and molecular mass spectrometry: its potential to assess phosphopeptide enrichment procedures.

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.

Study of phytochelatins and other related thiols as complexing biomolecules of As and Cd in wild type and genetically modified Brassica juncea plants.

The accumulation of As and Cd in Brassica juncea plants and the formation of complexes of these elements with bioligands such as glutathione and/or phytochelatins (PCs) is studied. The genetic manipulation of these plants to induce higher As and Cd accumulation has been achieved by overexpressing the genes encoding for gamma-glutamyl cysteine synthetase (gamma-ECS) and glutathione synthetase (GS). These two enzymes are responsible for glutathione (GSH) formation in plants, which is the first step in the production of PCs. The biomass produced in both the wild type and the genetically modified plants, has been evaluated. Additionally, the total Cd and As concentration accumulated in the plant tissues was measured by inductively coupled plasma mass spectrometry (ICP-MS) after extraction. Speciation studies on the extracts were conducted using size exclusion liquid chromatography (SEC) coupled online with ICP-MS to monitor As, Cd and S. For further purification of the As fractions, reversed phase high performance liquid chromatography (RP-HPLC) was used. Structural elucidation of the PCs and other thiols, as well as their complexes with As and Cd, was performed by electrospray-quadrupole-time-of-flight (ESI-Q-TOF). In both the Cd and As exposed plants it was possible to observe the presence of oxidized PC2 ([M + H]+, m/z 538), GS-PC2(-Glu) ([M + H]+, m/z 716) as well as reduced GSH ([M + H]+, m/z 308) and oxidized glutathione (GSSG) ([M + H]+, m/z 613). However, only the GS plants exhibited the presence of As(GS)3 complex ([M + H]+, m/z 994) that was further confirmed by MS/MS. This species is reported for the first time in B. juncea plant tissues.