Labelling of proteins with 2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid and lanthanides and detection by ICP-MS

Abstract

The labelling of two different proteins (bovine serum albumin, hen egg white lysozyme) with the commercially available chelating compound 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-SCN-Bn-DOTA) has been investigated. The assay described here has been optimised for the application to detect proteins labelled by stable isotopes of Eu, Tb and Ho and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Detection has been performed by laser ablation ICP-MS after electroblotting of the target proteins onto NC membranes. A range of total protein amounts from 0.015 pmol (BSA) to 105 pmol (lysozyme) has been covered. A calibration was performed for BSA in the range from 0.015 to 15 pmol and a limit of detection below 15 fmol can be estimated. For lysozyme integrated sensitivities of more than 10⁷ cps pmol⁻¹ of protein have been realized. The conditions, once optimised for labelling with Eu, have been applied for other lanthanides (Tb, Ho), too. ESI-MS of the intact and the tryptic digested lysozyme has been used as to provide a better understanding of the reaction chemistry and efficiency. The procedure described here looks promising to develop multielement labelling strategies (multiplexing) for ICP-MS applications in quantitative proteomics.