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This publication reflects the sum of work for the development of a quick and easy strategy for the determination of the protein relative concentration in individual human cells, by single cell–inductively coupled plasma–time-of-flight mass spectrometry (sc-ICP-ToF-MS). It included the incorporation of self-developed metal nanocluster (NC)-labeled specific antibodies for the target proteins. In addition, the cell samples were stained with ruthenium red (RR), a known membrane binding agent, and information about the number concentration and relative volume were also obtained. As a proof of concept, the expression of hepcidin, metallothionein-2, and ferroportin employing specific antibodies labeled with IrNCs, PtNCs, and AuNCs, respectively, was investigated by sc-ICP-ToF-MS in human ARPE-19 cells (human retinal pigment epithelial cells). Assuming that ARPE-19 cells are spherical in suspension and RR binds to the surface of the cells, the Ru intensity was corelated to the cell volume (i.e., the cell volume is directly proportional to, making it possible to determine not only the mass of the target proteins in each individual cell but also the relative concentration of the proteins.
The developped procedure will be of particular interest when comparing cell cultures subjected to different supplementations. During the study, ARPE-19 cell cultures under two stress conditions were compared: a hyperglycemic model and an oxidative stress model. The comparison of the control with treated cells shows not only the mass of analyzed species but also the relative changes in the cell volume and concentration of target proteins, clearly allowing the identification of subpopulations under the respective treatment.
Single Cell–ICP–ToF-MS for the Multiplexed Determination of Proteins: Evaluation of the Cellular Stress Response
Paula Menero-Valdés, Michail I. Chronakis, Beatriz Fernández*, C. Derrick Quarles Jr., Héctor González-Iglesias, Björn Meermann, and Rosario Pereiro
Published in Analytical Chemistry, Volume 95, Issue 35, Pages 13322-13329.
BAM Analytical Chemistry, Reference Materials
BAM Inorganic Trace Analysis