Single Cell–ICP–ToF-MS for the Multiplexed Determination of Proteins: Evaluation of the Cellular Stress Response

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.


Immunoassay with ARPE-19 Cells and MNCs-labelled Immunoprobes.
The immunoassays with MNCs-labelled immunoprobes were performed employing PBS as washing solution, Triton X-100 to permeabilize cellular membranes, and goat serum and bovine serum albumin to block non-specific interactions.After the immunoassay protocol, cells suspensions were diluted to 1•10 5 cells mL -1 in 50 mM Trizma® hydrochloride adjusting pH to 7.4 with hydrochloric acid.It is worth noting that our group previously conducted a study where different solvents (Milli-Q, NaCl, and Trizma) were investigated for ensuring RPE cells integrity when introducing them into the ICP.The experimental results revealed that the cellular transport efficiency with human RPE cells was significantly higher when using the Trizma buffer. 6The immunoassay protocols to label the three S4 target proteins in ARPE-19 cells were optimized in terms of immunoprobe concentration (referred to the antibody concentration) to ensure the total recognition of the proteins and the number of washing steps to avoid non-specific interactions.The protocols were independently performed with the three immunoprobes (Anti-h-HP:IrNCs, Anti-h-MT2:PtNCs or Anti-h-FPN:AuNCs).

sc-ICP-ToF-MS Analysis and Data Processing.
Table S1 collects the optimized operating conditions for the analysis of endogenous elements and MNCs-labels in ARPE-19 cells by sc-ICP-ToF-MS.
Table S1.sc-ICP-ToF-MS operating conditions.The instrument was tuned with STDS mode to measure Ru, Ir, Pt and Au.CCTS mode was employed for endogenous element detection.

Parameter Value
Sample flow rate (L min -1 ) 10 Plasma power (w) 1550 Gas flow rate (L min -  ARPE-19 cells were measured by microscopy (in suspension) to determine their dimensions.Cells were randomly selected from 30 images to measure their diameter, which was found to be 16 ± 4 µm (n=500).As an example, Figure S3 shows two optical images obtained for ARPE-19 cells.

S5Figure S1 .
Figure S1.Immunoassay optimization for the determination of HP, FPN and MT2 in CT ARPE-19 cells by sc-ICP-ToF-MS using Anti-h-HP:IrNCs, Anti-h-MT2:PtNCs and Anti-h-FPN:AuNCs immunoprobes.The protocol was independently performed with the three immunoprobes and different Ab concentrations were evaluated in each case.A) Hepcidin, B) Metallothionein-2, and C) Ferroportin.The graphs depict the concentration of the metal labels per cell.Uncertainties represent the standard deviation from the mean of three replicates.

 2  1 .
Figure S2 depicts the time resolved profile obtained for ARPE-19 cells after the immunoassay with MNCs-labelled immunoprobes and RR tagging by sc-ICP-ToF-MS.Two different types of events have been observed, denoted with numbers 1 and 2 in the Figure.Type 1 corresponds to cellular event where Ru, Ir, Pt or Au appeared individually, whereas Type 2 is associated to events where Ru was simultaneously detected with combinations of one, two or the three MNCs labels.

Figure S2 .
Figure S2.Time resolved profile obtained for ARPE-19 cells after the immunoassay with MNCslabelled immunoprobes and RR tagging by sc-ICP-ToF-MS.Two different type of events in the same cell suspension can be observed denoted as Type 1 (number 1 in the profile: Ru, Ir, Pt or Au appeared individually) and Type 2 (number 2 in the profile: Ru was simultaneously detected with combinations of one, two or the three MNCs labels).
3,2psin EDTA (Gibco, Thermo-Fischer Scientific) and fixated with paraformaldehyde (PFA; VWR Chemicals).The immunoassays with ARPE-19 cells in suspension using MNCs-labelled immunoprobes were performed employing phosphate buffered saline (0.1 M at pH 7.4) (PBS) as washing solution, triton X-100 (Sigma-Aldrich) to permeabilize cellular membranes and goat serum (Sigma-Aldrich) and bovine serum albumin (BSA; 99% powder, Merck) to block non-specific interactions.Cell membrane was tagged with ruthenium red (Sigma Aldrich).Trizma hydrochloride (99.9% crystals, Sigma-Aldrich) was used as buffer for sc-ICP-ToF-MS analyses.Transport efficiency for the sample introduction system was daily checked with a citrate stabilized PtNPs standard (46 ± 3 nm, NanoComposix).Ru, Ir, Au and Pt calibrations were performed with ICP S3 standards (1000 and culture time were selected based on previous studies with RPE cells in vitro.1,2Fourbiologicalreplicatesperconditionwere used: CT cells (both for GL and AAPH treatments), GL-treated cells and AAPH-treated cells.Cells analyzed by sc-ICP-ToF-MS were then subjected to a mild fixation with PFA and stored in PBS until further use.1.2.2.Synthesis and Labelling of Specific Antibodies with Metal Nanoclusters.IrNCs,PtNCs and AuNCs were synthesized and labelled with specific antibodies using previously optimized protocols.3-Takinginto account the amplification provided by each immunoprobe (i.e., the average number of metal atoms per Ab: 1760 for Ir, 1194 for Pt and 579 for Au) as well as the proteins concentration expected in RPE cells, Anti-h-HP was labelled with IrNCs, Anti-h-MT2 with PtNCs