QUANTITATION OF PHOSPHORUS IN BIOLOGICALLY ACTIVE MATERIALS BY ICP-MS DETECTION OF P AND S WITH CHEMICAL RESOLUTION

摘要

Protein phosphorylation and de-phosphorylation are key regulatory signalling processes that regulate critical biochemical pathways. These processes are catalysed by kinases and phosphatases, respectively. The activity of a kinase can be suppressed using a selective inhibitor, the synthesis of which is a crucial step in drug discovery. For example, a new class of anti-cancer drugs are inhibitors of an Epidermal Growth Factor Receptor (EGFR), which inactivates its intracellular tyrosine kinase activity [1]. Screening of such drug candidates necessitates assessment of the phosphorylation state of a potential protein target. Typically, a Western blot is probed with phospho-specific antibodies in order to obtain a profile of the phospho-proteins separated by gel electrophoresis. A digital answer of the presence of a phosphorylated protein is obtained. Recently, a phosphoELISA~(TM) kit for 96-well format became available that utilises an immuno-precipitation assay with colorimetric detection of phospho-specific antibody conjugates [2]. A separate ELISA is performed for total protein quantitation in order to determine phosphorylation levels. The ELISA-based methods are the most sensitive and can attain as low as 5 pg/mL detection levels for some proteins [2]. The phosphorylation state of a protein can potentially be determined by detection of total phosphate in the purified protein sample. The most common analytical method for detection of total phosphate is molybdenum blue with stannous chloride reduction of molybdophosphoric acid [3] with detection range of about 1 ng/mL to 10 (mu)g/mL. Another common, sensitive, but hazardous method used in biochemistry is scintillation detection of ~(32)P-radioisotope [4]. On the other hand, ICP-MS, which has been successfully applied for detection of phosphorus [5-10], offers the advantage of simultaneous detection of other elements. In most cases, metals bound to the biomolecules of interest are detected. Recently, detection of sulfur has been shown to be useful for compensation in variability of total sample amounts [9,10]. Polyatomic isobaric interferences on ~(32)S~(+) and ~(31)P~(+) are resolved either by employing high resolving power of sector field ICP-MS [9], or by chemical resolution in ICP-DRC-MS [10].