Unveiling compositional images of specific proteins in individual cells by LA-ICP-MS: Labelling with ruthenium red and metal nanoclusters

• Published in: Analytica chimica acta 2024-08, Vol.1317, p.342906, Article 342906
• Main Authors: Menero-Valdés, Paula, Álvarez, Lydia, González-Iglesias, Héctor, Fernández, Beatriz, Pereiro, Rosario
• Format: Article
• Language: English
• Subjects: Antibody labelling; Cell Line; Cytochrome P-450 CYP1B1 - metabolism; Cytosolic proteins; Humans; Laser ablation ICP-MS; Mass Spectrometry; Metal nanoclusters; Metal Nanoparticles - chemistry; Oxidative Stress; Quantitative distribution of proteins
• ISSN: 0003-2670; 1873-4324; 1873-4324
• DOI: 10.1016/j.aca.2024.342906

Recent biological studies have demonstrated that changes can occur in the cellular genome and proteome due to variations in cell volume. Therefore, it is imperative to take cell volume into account when analyzing a target protein. This consideration becomes especially critical in experimental models involving cells subjected to different treatments. Failure to consider cell volume could obscure the studied biological phenomena or lead to erroneous conclusions. However, quantitative imaging of proteins within cells by LA-ICP-MS is limited by the lack of methods that provide the protein concentration (protein mass over cell volume) rather than just protein mass within individual cells. The combination of a metal tagged immunoprobe with ruthenium red (RR) labelling enables the simultaneous analysis of a specific protein and the cell volume in each cell analyzed by LA-ICP-(Q)MS. The results indicate that the CYP1B1 concentration exhibits a quasi–normally distribution in control ARPE-19 cells, whereas AAPH–treated cells reveal the presence of two distinct cell groups, responding and non–responding cells to an in vitro induced oxidative stress. The labelling of the membrane with RR and the measurement of Ru mass in each cell by LA-ICP-MS offers higher precision compared to manually delimitation of the cell perimeter and eliminates the risk of biased information, which can be prone to inter–observer variability. The proposed procedure is fast and minimizes errors in cell area assignment and offers the possibility to carry out a faster data treatment approach if just relative volumes are compared, which can be advantageous for specific applications. This work presents an innovative strategy to directly study the distribution and concentration of proteins within individual cells by LA-ICP-MS. This method employs ruthenium red as a cell volume marker and Au nanoclusters (AuNCs) tagged immunoprobes to label the protein of interest. Furthermore, the proposed labelling strategy enables rapid data processing, allowing for the calculation of relative concentrations and thus facilitating the comparison across large datasets. As a proof–of–concept, the concentration of the CYP1B1 protein was quantified in ARPE-19 cells under both control and oxidative stress conditions. [Display omitted] •Concentration of proteins within individual cells was obtained by LA-ICP-MS.•The simultaneous analysis of a specific protein and the cell volume was achieved.•Ruthenium red was employed a