Determination of proteins in hypoxic Müller glia cells by single-cell ICP-MS: The use of a Rh-DNA intercalator for enhanced cell detection

• Concentration of proteins within individual cells was obtained by sc-ICP-QMS. • Endogenous elements and two external tags were evaluated for cell detection. • Rh-DNA intercalator improves discrimination of cell events by sc-ICP-QMS. • AuNCs and IrNCs were used as specific antibody labels to develop metal immunoprobes. • MIO-M1 cell populations, subjected to hypoxia conditions or to normoxia, were studied. Single cell − inductively coupled plasma-mass spectrometry (sc-ICP-MS) enables the identification and quantification of elements and biomolecules within individual cells. Target proteins can be analyzed by sc-ICP-MS using metal nanoclusters (MNCs)-based immunoprobes, facilitating the detection of low amounts of proteins in each cell. However, for the full implementation of sc-ICP-MS, an effective strategy to enhance the detection of cellular events is required. In this work, the measurement of endogenous elements and two external tags (ruthenium red and Rh-DNA intercalator) was investigated for cell detection. As a proof of concept, the sequential determination of two proteins (hypoxia inducible factor-1α and vascular endothelial growth factor; HIF-1α and VEGF, respectively) in individual human Müller glia cells (MIO-M1) was pursued. Specific antibodies against HIF-1α and VEGF were labelled with AuNCs and IrNCs, respectively. The results demonstrated the advantages of using Rh-DNA intercalator for the detection and discrimination of cellular events by sc-ICP-MS for quadrupole (Q) mass spectrometers. Additionally, applying this analytical method to cultured MIO-M1 cells under hypoxic and to normoxic conditions showcased the ability of sc-ICP-QMS to study hypoxia-induced changes in the cell-to-cell distributions of HIF-1α and VEGF levels.