Quantification of Protein Phosphorylation by μLC-ICP-MS

Abstract

Determination of the protein amount and of the extent of protein phosphorylation is crucial for a variety of research fields, but is not always straightforward. We describe the application of capillary LC-ICP-MS (liquid chromatography-inductively coupled plasma-mass spectrometry) for quantification of phospho-proteins and their phosphorylation degree. Element mass spectrometry is ideally suited for monitor ing and quantification of compounds with heteroelements such as phosphorus and sulphur, particularly because the ICP-MS response is virtually independent from the chemical form of the element. Determination of the phosphorylation stoichiometry, i.e. the relative abundance of the phosphorylated isoforms, can be assessed by the relative abundance of phosphorus compared with sulphur as a marker for the protein amount. Moreover, isotope dilution analysis by post-column addition of a 34S-Spike provides absolute protein quantification with exceptionally high accuracy. Phosphoprotein analysis by capillary LC-ICP-MS may be applied to isolated proteins or protein digests and may include separation of impurities by 1D-SDS-PAGE followed by enzymatic digestion. Alternatively, digestion of complex protein mixtures such as cellular protein extracts allows determination of global, tissue-specific phosphorylation degrees.