No abstract

No abstract

No abstract

An automated and straightforward detection and data treatment 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) is proposed. Metal nanocluster (NC)-labeled specific antibodies for the target proteins were employed, and ruthenium red (RR) staining, which binds to the cells surface, was used to determine the number of cell events as well as to evaluate the relative volume of the cells. 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. Taking into account that ARPE-19 cells are spherical in suspension and RR binds to the surface of the cells, the Ru intensity was related to the cell volume (i.e., the cell volume is directly proportional to (Ru intensity) 3/2 ), making it possible to determine not only the mass of the target proteins in each individual cell but also the relative concentration. The proposed approach is of particular interest in comparing cell cultures subjected to different supplementations. 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.

The use of metal immunoprobes, defined as recognition molecules (e.g., antibodies) labeled with metal tags, constitutes an interesting strategy for the analysis of proteins in biological samples. Fluorescent and biocompatible metal nanoclusters (MNC) have been recently established as powerful tags for detection by spectrofluorimetry, but also by elemental mass spectrometry (MS). Detection of such immunoprobes by elemental MS allows not only the qualitative analysis of the proteins but also their absolute quantification. However, the deviation associated with the MNCs polydispersity will limit the analytical precision, particularly in those samples where the concentrations of the sought protein are very low (e.g., single cell analysis). In this work the synthesis of size monodisperse gold nanoclusters (AuNCs) is investigated by using different experimental conditions such as reaction time and temperature, solvent, reducing agent, and pH, among others. Characterization of AuNCs was performed by spectrofluorimetry, dynamic light scattering (DLS) and high resolution transmission electron microscopy (HR-TEM) measurements.