Absolute and Site-Specific Quantification of Protein Phosphorylation Using Integrated Elemental and Molecular Mass Spectrometry: Its Potential To Assess Phosphopeptide Enrichment Procedures
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- 作者：Ana Pereira Navaza ; Jorge Ruiz Encinar ; Montserrat Carrascal ; Joaquí ; n Abiá ; n ; Alfredo Sanz-Medel
- 刊名：Analytical Chemistry
- 出版年：2008
- 出版时间：March 1, 2008
- 年：2008
- 卷：80
- 期：5
- 页码：1777 - 1787
- 全文大小：186K
- 年卷期：v.80,no.5(March 1, 2008)
- ISSN：1520-6882
文摘
The validity of using elemental phosphorus standards toaccurately and precisely quantify phosphopeptides bycapillary HPLC (capHPLC) coupled to ICP-collison cell-MS is investigated in detail. Operating requirements tomaintain stable 31P sensitivity along the reversed-phasegradient are described. Specifically, the use of an optimum postcolumn makeup flow with a defined acetonitrilecontent turned out to be necessary to buffer the acetonitrile variation of the capillary chromatographic eluent andensure plasma stability. Then, a highly pure P-containingstandard (bis(4-nitro-phenyl) phosphate, BNPP) wasspiked into the samples and used to quantify them withvery low absolute errors (2-4%) and excellent precision(3-6%). The capHPLC-ICPMS method showed excellentlinearity over 3 orders of magnitude and provided adequate detection limits (110 fmol, 3.4 pg P). Accuratequantification of the phosphopeptides present in a trypticdigest of 
-casein and casein from bovine milk was thenattempted. Previously, and in order to be able to closemass balances, total P contents, percentages of inorganicP present, and recoveries from the reversed-phase column used in the separation were computed for eachsample. Quantification using the spiked BNPP for thedifferent phosphopeptides detected matched the expectedvalues well validating the quantitative methodology proposed. The capHPLC-ESIMS analysis allowed elucidatingamino acid sequences, a requisite still necessary totranslate the determined amount of P in each chromatographic peak into amount of phosphopeptide. The greatpotential of these strategies, based on ICPMS detection,to assess the many procedures proposed and commonlyused for purification, preconcentration, and/or isolationof phosphopeptides in phosphoproteomics studies isdemonstrated using a commercially available titaniumdioxide (TiO2) cartridge for phosphopeptide enrichmentfrom complex mixtures. Quantitative results obtainedallow one to assess individual phosphopeptide recoveriesfrom the TiO2 cartridge with unsurpassed accuracy. Ofcourse, this information is essential for reliable absolutequantifications in phosphoproteomics.
-casein and casein from bovine milk was thenattempted. Previously, and in order to be able to closemass balances, total P contents, percentages of inorganicP present, and recoveries from the reversed-phase column used in the separation were computed for eachsample. Quantification using the spiked BNPP for thedifferent phosphopeptides detected matched the expectedvalues well validating the quantitative methodology proposed. The capHPLC-ESIMS analysis allowed elucidatingamino acid sequences, a requisite still necessary totranslate the determined amount of P in each chromatographic peak into amount of phosphopeptide. The greatpotential of these strategies, based on ICPMS detection,to assess the many procedures proposed and commonlyused for purification, preconcentration, and/or isolationof phosphopeptides in phosphoproteomics studies isdemonstrated using a commercially available titaniumdioxide (TiO2) cartridge for phosphopeptide enrichmentfrom complex mixtures. Quantitative results obtainedallow one to assess individual phosphopeptide recoveriesfrom the TiO2 cartridge with unsurpassed accuracy. Ofcourse, this information is essential for reliable absolutequantifications in phosphoproteomics.