Quantification of elemental labeled peptides in cellular uptake studies

A methodological study on peptide quantification was carried out comparing LC-ESI-TOFMS with LC-ICP-MS approaches. For this purpose a peptide without hetero-elements amenable to ICP-MS analysis has been labeled by indium using 1,4,7,10-tetraazacyclododecane-N, N', N, N'-tetraacetic acid as complexing tag (In-DOTA). Accordingly, quantification of the free labeled compound was carried out by LC-ICP-MS and compared to LC-ESI-TOFMS measurement without labeling, applying identical chromatographic conditions. For aqueous standards both methods showed comparable results in terms of sensitivity and limit of detection (1.7 and 3.2 fmol, respectively). In cell culture experiments, endothelial cells were incubated with the labeled and unlabeled peptide for 5 minutes. Immunohistochemistry on fixed cells revealed cytosolic fluorescence indicating internalization of the peptide. To quantify the amount of peptide uptake, cytoplasm was isolated via hyperosmolar cell lysis and either directly analyzed or subjected to different sample preparation techniques to obtain the low molecular mass fraction. Peptide concentrations were determined by LC-ICP-MS and LC-ESI-TOFMS. Despite the comparable limits of detection in aqueous samples, measurement of the cytoplasm samples revealed severe matrix effects in the case of LC-ESI-MS making quantification at the present concentration levels impossible. In this respect a clear advantage of LC-ICP-MS quantification of peptides in combination with elemental labeling was demonstrated.