Elemental mapping of biological samples by the combined use of LIBS and LA-ICP-MS

Feasibility of the simultaneous use of LIBS and LA-ICP-MS (tandem LA/LIBS) for bio-imaging of bulk and trace elements is demonstrated.</p

Analysis of Individual Cells by ICP-MS: New Analytical Strategies Using Laser Ablation and Single-Cell ICP-MS for the Determination of Biomolecules

Trabajo presentado en el Advances in Atomic Spectroscopy, celebrado online los días 28 de febrero y 1 de marzo de 202

Determination of Endogenous Proteins in Individual Cells From an In Vitro Model by Elemental Mass Spectrometry

Trabajo presentado en el 1st PhD Multidisciplinary Chemical Congress, celebrado en Gijón (España), los días 19 y 20 de enero de 2023Cell populations in biological systems are well-known for their heterogenous nature. Under the same physiological conditions and external stimuli, cells from the same line may show different biomolecule expression. For that reason, differences among cell populations can be difficult to elucidate unless they are studied cell by cell. In this context, there is a need to develop new analytical methodologies allowing for the quantitative determination of the content (endogenous metals and biomolecules) of individual cells. Over the last few years, single cell (sc) ICP-MS has been employed for the analysis of the endogenous metal content of individual cells, the uptake of metallodrugs or the internalization of metallic nanoparticles. Moreover, if combined with immunocytochemistry, it is possible also to determine endogenous biomolecules within individual cells if labelled with metal tagged immunoprobes [1]. However, spatial information is lost with this sample introduction strategy. This could be overcome by sampling the cells using laser ablation (LA) coupled to ICP-MS. In this work, sc-ICP-MS and LA-ICP-MS are investigated as complementary tools for a comprehensive study of specific proteins in individual cells from an in vitro model of retinal pigment epithelium (ARPE-19 cell line). On the one hand, sc-ICP-MS allows for the quantitative determination of proteins in single cells even at attogram level [2]. On the other hand, LA-ICPMS allows the elucidation of the protein spatial distribution within cellular structures. The methodology employed for the study of specific proteins in ARPE-19 cells uses iridium nanoclusters (IrNCs) as the elemental label for ICP-MS detection. A case study for the determination of APOE and Claudin-1 proteins in individual ARPE-19 cells (control and subjected to a stress treatment with glucose) is presented.This work was financially supported through projects PID2019- 107838RB-I00/Agencia Estatal de Investigación (AEI)/10.13039/ 501100011033) in Spain and AYUD/2021/51289 - FICYT. P. Menero-Valdés acknowledges the FPU Grant with Ref. FPU19/00556 (Ministry of Education, Spain)

Identification of trace elements and biomolecules in extracellular vesicles secreted by human RPE cells by ICP-MS

Resumen del trabajo presentado 19th European Winter Conference on Plasma Spectrochemistry, celebrado en Ljubljana (Eslovenia), del 29 de enero al 3 de febrero de 2023Age-related macular degeneration (AMD) is one of the leading causes of irreversible blindness, affecting almost 200 million people worldwide. Previous studies of AMD have revealed that progressive degeneration of the retinal pigment epithelial (RPE) cells play an important role in the development of AMD. Human RPE cells secrete extracellular vesicles (EVs), whose cell-tocell signalling function may be crucial during early stages of AMD (1). Therefore, the study of the possible implication of EVs in dyshomeostasis and RPE cell degeneration during AMD is of great research interest.EVs are nano-sized vesicles containing nucleic acid and protein cargo that are released from a multitude of cell types and have gained significant interest as potential diagnostic biomarkers. Biological fluids (including cell culture media, urine, plasma and serum) are rich sources of readily accessible EVs. However, the isolation of EVs from proteins and non- EV lipid particles represents a considerable challenge nowadays.In this work we present the development of protocols for the purification of EVs from cell culture media of in vitro models of human RPE cells (hRPE), subjected to a pro-oxidative stress treatment and control, for the determination of trace metals and proteins in EVs. Two isolation strategies were evaluated by differential centrifugation and precipitation. The characterization of purified EVs was further performed by dynamic light scattering, nanoparticle tracking analysis, and transmission electron microscopy. Concerning metals determination, and taking into account the limited sample volume (less than 100 L), different sample introduction systems were evaluated to obtain Fe, Cu and Zn concentration in EVs by ICP-MS. Metal nanoclusters (MNCs) were employed as the elemental label for CD81 and TSG101 proteins detection by ICP-MS.EVs analysed by conventional nebulization ICP-MS provides information of the whole population as an average. However, it is well-known that cells in all biological systems have a heterogeneous nature and, therefore, EVs can also exhibit a non-homogeneous behaviour. The combination of single cell (sc) ICP-MS with an immunocytochemistry step using MNCs has been explored to identify EVs purified from control and treated individual hRPE cells

Determination of target proteins in immortalized and primary RPE cells by sc-ICP-MS using metal nanoclusters as labels

Resumen del trabajo presentado 19th European Winter Conference on Plasma Spectrochemistry, celebrado en Ljubljana (Eslovenia), del 29 de enero al 3 de febrero de 2023Traditionally, cellular samples have been analysed by conventional nebulization ICP-MS after digestion of the cell, providing information of the whole population as an average (i.e., information regarding individual heterogeneity is lost). However, it is well known that cell populations have a heterogeneous nature and, thus, the study of cell populations on a cell-tocell basis is frequently required. The determination of the elemental composition of individual cells can be successfully achieved employing single cell (sc) ICP-MS (1). This technique allows the quantitative study of endogenous elements in cell populations. Furthermore, the combination of sc-ICP-MS with immunocytochemistry makes it possible to determine proteins in individual cells. In this case, the cellular samples are subjected to an immunoassay using an elemental label for detection (e.g., DOTA, MAXPAR, or nanoparticles). In this work, a biomarker strategy employing metal nanoclusters (MNCs) as elemental labels for the sequential determination of membrane and cytosolic proteins in individual human retinal pigment epithelial (RPE) cells was performed by sc-ICP-MS. The proposed methodology is based on immunoassays in cell suspension using MNCs conjugated to protein specific antibodies for protein determination in individual RPE cells (ARPE-19 and primary human fetal RPE cell lines). For such purpose, NCs of two different metals were evaluated as labels: AuNCs and IrNCs. The metal to antibody stoichiometry in the Au and Ir labelled immunoprobes was determined in order to provide quantitative determination of the target proteins in individual cells.This work was financially supported through project AYUD/2021/51289 (PCTI Program of the Government of the Principality of Asturias and FEDER Program of the European Union) and PID2019-107838RB-I00/Agencia Estatal de Investigación (AEI)/10.13039/501100011033) in Spain. P. Menero-Valdés acknowledges the FPU Grant with Ref. FPU19/00556 (Ministry of Education, Spain)

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.This work was financially supported through projects PID2019-107838RB-I00/Agencia Estatal de Investigación (AEI)/10.13039/501100011033) and AYUD/2021/51289- FICYT. P. Menero-Valdés acknowledges the FPU Grant with ref FPU19/00556 (Ministry of Education, Spain). The authors acknowledge Charlie Tobias (Federal Institute for Materials Research and Testing, Division 1.1 − Inorganic Trace Analysis) for the cytometry measurements and Lyndsey Hendricks (Tofwerk) for the technical support with the software used for data treatment.Peer reviewe

Bioimaging of specific proteins by single cell laser ablation ICP-MS employing iridium nanoclusters as elemental labels

Resumen del trabajo presentado en el 15th European Workshop on Laser Ablation (EWLA 2022), celebrado en Berna (Suiza), del 12 al 15 de julio de 2022Cell populations in all biological systems are well-known for their heterogeneous nature. In fact, individual cells of the same line, under same physiological conditions and external stimuli, may show different biomolecule expression. Therefore, differences among cell populations can be difficult to elucidate unless biological systems are studied on a cell-by-cell basis. At present, there is a need for innovative analytical techniques that allow for the determination of elements and biomolecules in individual cells. In this work, LA-ICP-MS and single cell (sc) ICP-MS are presented as complementary strategies for the determination of specific proteins in cell cultures. On the one hand, LA-ICP-MS has demonstrated a huge potential for determining the spatial distribution of metals in individual cells. Although the bioimaging of endogenous cellular proteins still remains a challenge, an immunoassay in fixated cells using immunoprobes conjugated with an elemental label can be employed for proteins detection. On the other hand, sc-ICP-MS has been proposed for high-throughput cell-by-cell quantitative determination of target proteins within cell cultures [1]. Therefore, combining these two techniques it is possible to determine the concentration of sought proteins in single cells as well as to obtain their spatial distribution along the cell structures. In the proposed methodology, well characterized small iridium nanoclusters (IrNCs) providing a high signal amplification were employed as labels for the bioimaging of membrane and cytosolic proteins in individual retinal pigment epithelia cells (ARPE-19 cell line) by LA-ICP-MS (NWR193 laser equipped with a TwoVol2 ablation cell and DCI interface coupled to a 7900 ICP-MS). Protein quantification in individual cells was studied by sc-ICP-MS (microFAST Single Cell system coupled to a 7900 ICP-MS) and the proteins distribution was evaluated by LA-ICP-MS. The novel approach was based on immunoassays (cells fixed on chamber slides for LA-ICP-MS and cell suspensions for sc-ICP-MS) using IrNCs conjugated to specific antibodies for determination of the sought proteins under control and stress conditions (glucose treatment, 100 mM).This work was financially supported through project PID2019-107838RB-I00 Agencia Estatal de Investigación (AEI)/10.13039/501100011033) and project AYUD/2021/51289 - FICYT. P. Menero- Valdés acknowledges the FPU Grant (Ref. FPU19/00556; Ministry of Education)

Metal nanoclusters as elemental labels for the determination of specific proteins related to eye neurodegenerative diseases in individual cells by single cell and laser ablation ICP-MS

Resumen del trabajo presentado en el Colloquium Spectroscopicum Internationale XLII (CSI XLII), celebrado en Gijón (España), del 30 de mayo al 3 de junio de 202

Determination and localization of specific proteins in individual ARPE-19 cells by single cell and laser ablation ICP-MS using iridium nanoclusters as label

Single cell-inductively coupled plasma-mass spectrometry (sc-ICP-MS) and laser ablation (LA)-ICP-MS have been complementary employed to develop a comprehensive study of APOE and claudin-1 expression in ARPE-19 cells submitted to a glucose treatment (100 mM, 48 h) that induces oxidative stress conditions. Results were compared with control cells. The determination of the two proteins by ICP-MS was sequentially carried out using specific immunoprobes labelled with IrNCs that offer a huge amplification (1760 ± 90 atoms of Ir on average). A novel sample introduction system, the microFAST Single Cell set-up, was employed for sc-ICP-MS analysis. This introduction system resulted in a cellular transport efficiency of 85 ± 9% for ARPE-19 cells (91 ± 5% using a PtNPs standard). After the proper immunocytochemistry protocol with the specific IrNCs immunoprobes in cell suspensions (sc-ICP-MS), the mass of APOE and claudin-1 in individual ARPE-19 cells was obtained. Average detection limits per cell by sc-ICP-MS were 0.02 fg of APOE and 3 ag of claudin-1. The results of sample analyses obtained by sc-ICP-MS were validated with commercial ELISA kits. The distribution of both target proteins in individual cells (fixated in the chamber wall) was unveiled by LA-ICP-MS. The high amplification provided by the IrNCs immunoprobes allowed the identification of APOE and claudin-1 within individual ARPE-19 cells. High resolution images were obtained using a laser spot of 2 × 2 μm.This work was financially supported through projects PID2019- 107838RB-I00/Agencia Estatal de Investigacion ´ (AEI)/10.13039/ 501100011033) in Spain and AYUD/2021/51289 - FICYT. P. MeneroVald´es acknowledges the FPU Grant with Ref. FPU19/00556 (Ministry of Education, Spain). Authors would like to acknowledge David Clases (University of Graz, Austria) for the support with SPCaL software and the technical support provided by Servicios Científico-T´ecnicos of the University of Ovied