Detection of phosphorylated proteins blotted onto membranes using laser ablation inductively coupled plasma mass spectrometry Part 1: Optimisation of a calibration procedure

Abstract

Inductively coupled plasma mass spectrometry (ICP-MS) has been used already for biological applications, especially for determination of the phosphorylation status of proteins. For this purpose we have coupled a laser ablation (LA) system to ICP-MS for detection of hetero-elements on membranes after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electro-blotting. Our experiments show good reproducibility with a standard deviation of 6.1%. In single line scans nitrocellulose membrane material showed significantly higher signal intensities in comparison to polyvinylidene fluoride membranes. Quantification of phospho-proteins has been performed with (1) dotting and (2) SDS-PAGE separated and blotted standards to overcome protein losses during blotting. With this method, good linearity could be achieved in the range from 20 to 500 pmol phosphorus in proteins. We have estimated the limit of detection of phosphorus in β-casein, evaluating the whole protein spot to be 1.5 pmol using the 3σ criterion measured in a medium resolution of the sector field instrument.