No 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 heteroelements 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 b-casein, evaluating the whole protein spot to be 1.5 pmol using the 3s criterion measured in a medium resolution of the sector field instrument.

Numerous structurally and enzymatically similar cytochromes P450 (CYPs) are involved in the metabolism of xenobiotics and are present in different amounts and with different enzyme profiles in human tissues and cells. Analysis of their adaptively regulated and individually variable patterns is a peculiar analytical challenge. We developed a laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) based method for concomitant detection and semiquantitative determination of electrophoretically separated and blotted CYPs. The first results are given here for the two enzymes CYP1A1 and CYP2E1. Specific monoclonal antibodies directed against the enzymes were differentially labelled with europium via a covalently linked chelator and with iodine, respectively. Analysis of the modified antibodies shows that both europium and iodine are coupled to the heavy and the light chains of the antibodies. Also, the antibodies maintained their antigen-binding properties after labelling as demonstrated by LA-ICP-MS-analysed immunoblots. The method allowed us to detect specifically and concomitantly both CYP enzymes in complex biological samples, i.e. microsomes of rat liver and minipig duodenum, which are characterized by different levels and proportions of the two CYP enzymes. A strong CYP1A1 signal is found in liver microsomes of 3-methylcholanthrene-treated rats, while it is (nearly) absent in liver microsomes of rats treated with isonocotinic acid hydrazide (isoniazid). The constitutively expressed CYP2E1 is found in microsomes of both treatment groups. Duodenal microsomes of minipigs orally exposed to polycyclic aromatic hydrocarbons show a clear CYP1A1 signal. Low levels of CYP2E1 can also be detected in these microsomes. The LA-ICP-MS method allows concomitant determination of CYPs, thereby exhibiting sensitivity similar to that of conventional chemoluminescence detection via peroxidase-labelled secondary antibodies. The latter method allows readout of a single CYP protein in a 1D separation. Although the results presented here are only for labelling by use of the elements iodine and europium, the same strategy can be applied also for other lanthanide elements in combination with chelating compounds, so LA-ICP-MS of western blots offers a new capability to be applied for highly multiplexed CYP determinations via labelled antibodies.