Determination and localization of specific proteins in individual ARPE-19 cells by single cell and laser ablation ICP-MS using iridium nanoclusters as label

Abstract

Single cell-inductively coupled plasma-mass spectrometry (sc-ICP-MS) and laser ablation (LA)-ICP-MS have been complementary employed to develop a comprehensive study of APOE and claudin-1 expression in ARPE-19 cells submitted to a glucose treatment (100 mM, 48 h) that induces oxidative stress conditions. Results were compared with control cells. The determination of the two proteins by ICP-MS was sequentially carried out using specific immunoprobes labelled with IrNCs that offer a huge amplification (1760 ± 90 atoms of Ir on average). A novel sample introduction system, the microFAST Single Cell set-up, was employed for sc-ICP-MS analysis. This introduction system resulted in a cellular transport efficiency of 85 ± 9% for ARPE-19 cells (91 ± 5% using a PtNPs standard). After the proper immunocytochemistry protocol with the specific IrNCs immunoprobes in cell suspensions (sc-ICP-MS), the mass of APOE and claudin-1 in individual ARPE-19 cells was obtained. Average detection limits per cell by sc-ICP-MS were 0.02 fg of APOE and 3 ag of claudin-1. The results of sample analyses obtained by sc-ICP-MS were validated with commercial ELISA kits. The distribution of both target proteins in individual cells (fixated in the chamber wall) was unveiled by LA-ICP-MS. The high amplification provided by the IrNCs immunoprobes allowed the identification of APOE and claudin-1 within individual ARPE-19 cells. High resolution images were obtained using a laser spot of 2 × 2 μm.