Determination of Proteins in Single Cells by Inductively Coupled Plasma–Mass Spectrometry Using Metal Nanoclusters as Labels of Specific-Recognition Reactions

The research interests of Beatriz Fernandez, who is an Associate Professor at the University of Oviedo in Spain, are mainly focused on the field of spatially resolved analysis by mass spectrometry-based techniques, particularly glow discharge (GD) sources and laser ablation inductively coupled plasma-mass spectrometry (ICP-MS). Fernandez’s research is mainly focused on imaging studies of biomedical interest tissues and cell cultures for detection of heteroatoms and proteins, with special efforts devoted to development of accurate strategies for absolute quantification, including the use of stable isotopically-enriched tracers (isotope dilution MS). Recently, at the 2022 SciX conference in Covington, Kentucky, Fernandez presented, as part of the Technical Program, a discussion regarding the determination of proteins in single cells by ICP-MS using metal nanoclusters as labels of specific recognition reactions. Fernandez spoke to Spectroscopy about her presentation.

What makes inductively coupled plasma-mass spectrometry (ICP-MS) an essential tool for quantitative ultratrace elemental and isotopic determinations in biological samples?

Nowadays ICP-MS is a well-established technique, both in research and routine laboratories, for analyzing very low concentrations of metallic and semi-metallic elements in a wide variety of samples, including biological samples. ICP-MS does not only allow for the detection of the elements present in the sample (their identification), but also allows for the determination of accurate and precise concentrations of the target analytes (their quantification). Compared to traditional techniques employed in clinical or biological laboratories, the possibility to achieve the determination of absolute concentrations of ultratrace elements makes the ICP-MS technique extremely attractive for the analysis of biological samples. Additionally, the analysis of liquid samples (including, but not limited to, blood, urine, and aqueous humor) can be done in an almost straightforward manner with minimum sample preparation. For the analysis of other type of samples, such as tissue sections or cultured cells, a previous digestion step is required for the analysis by conventional nebulization ICP-MS. However, we can find alternatives by using ICP-MS coupled to single cell (sc) or laser ablation (LA) for the direct analysis of individual cells (without the prior digestion).

How is measuring proteins in single cells related to elemental analysis using ICP-MS?

Due to the high temperature reached within the ICP and the atomization of the sample, ICP-MS technique can only be used to get elemental information from the samples. Therefore, for the analysis of target proteins in single cells, it is necessary to resort to an immunoassay using specific antibodies for the proteins of interest (antigen:antibody recognition reaction). Additionally, such antibodies have to be labeled with an easily detectable element by ICP-MS (not present in the native cells). Thus, it can be stated that elemental analysis in single cells is simpler compared to the analysis of proteins since the immunocytochemistry step is not required. In any case, it should be highlighted that for the analysis of single cells by ICP-MS is necessary to use sample introduction systems fit to purpose: for example, sc-ICP-MS to detect individual cell events in a cell suspension or LA-ICP-MS to identify the inner distribution of individual cells.

What are metal nanoclusters and how are they used to study specific immunochemical reactions?

Recent advances in nanotechnology have introduced a new class of nanoparticles, named as metal nanoclusters (MNCs). MNCs have typical sizes between 0.2 and 3 nm. This type of nanostructure is characterized for having sizes comparable to the Fermi wavelength of electrons and, therefore, they can exhibit molecule-like properties (fluorescence). In addition, MNCs are composed of a few to several hundred atoms of a unique metal, providing high signal amplification by MS detection. Concerning how are they used to study specific immunochemical reactions, the surface of the nanostructure can be tailored with selected groups for different chemistries. For example, carboxylic groups of the MNCs surface can be bioconjugated with specific primary antibodies (for the target proteins) by carbodiimide coupling to act as a label immunoprobe.

How do metal nanoclusters compare to other labeling techniques as metal tagged immunoprobes?

Recently there has been a burgeoning interest in applying water soluble MNCs for biomedical and biosensing applications. MNCs present excellent properties compared to alternative labels, such as metal chelates like tetraxetan (DOTA), polymers containing several metal chelates, quantum dots, or metal nanoparticles—mainly due to their combination of small volume and high number of metal atoms per label. On the one hand, their small size (below 3 nm) makes them of interest for labeling biomolecules such as antibodies, since they will have a lower risk of hindering the antibody recognition capabilities compared with larger tags. On the other hand, the high number of metal atoms per label (up to 1000 metal atoms can be possible per NC depending on the MNCs) provides a high amplification by MS detection. Finally, it can be also pointed out that their fluorescent emission as well as their catalytic properties offer the possibility of multimodal detection—by fluorescence, electrochemical techniques, and by MS detection.

What has been learned so far using metal nanoprobes to study immunochemical reactions in human cells?

The use of metal nanoprobes, such as MNCs, offers us the possibility of achieving molecular information by using the ICP-MS elemental technique. Furthermore, the high sensitivity of ICP-MS together with the high amplification provided by metal nanoprobes make affordable the analysis of proteins in individual cells at very low concentrations. For example, specific proteins in the attogram level can be detected and quantified in individual cells by using sc-ICP-MS and LA-ICP-MS. ICP-MS also shows another important feature for the analysis of human cells: by using state-of-the-art instrumentation (for instance, time of flight mass spectrometry) and several metal labeled nanoprobes it is possible to obtain the simultaneous analysis of different target biomolecules in individual cells.

What are the challenges or limitations involved with using this technique?

Here we can distinguish between the determination of proteins in individual cells by sc-ICP-MS or LA-ICP-MS. In both cases, the accurate and precise determination of the immunoprobe stoichiometry (such as the number of metal atoms per antibody molecule that will be detected by MS) is mandatory. Concerning sc-ICP-MS one of the main challenges is associated with the sample introduction system: a high transport efficiency is required to get representativeness of the cell population as well as preserving cell integrity during the transport to the ICP. On the other hand, the determination of protein concentration by LA-ICP-MS is also challenging, considering the lack of certified reference materials for biological samples. Laser-based techniques exhibit matrix effects and, therefore, calibration standards with a similar matrix to the sample matrix is required to get accurate and precise protein concentrations.

As experience with the technique advances, and mastery of the technique is achieved, what are the expected advances for clinical research discovery?

It is well known that cell populations in all biological systems are heterogeneous. Furthermore, genome, transcriptome, and proteome cell-to-cell variations can be the origin of different pathologies. The quantitative analysis of individual cells in a target population is currently a critical research area in order to evaluate cellular variability as well as to study the behavior of cells submitted to different external stimuli (for example, applicability of treatments to specific diseases). Traditionally, cellular samples have been analyzed by conventional nebulization ICP-MS after digestion of the cells, providing information of the whole population as an average (so, information regarding individual heterogeneity is lost). The combination of sc-ICP-MS and LA-ICP-MS for the analysis of individual cells allows a comprehensive quantitative study of the expression of endogenous proteins by cell populations.

Beatriz Fernandez

Beatriz Fernandez is Associate Professor in the Department of Physical and Analytical Chemistry at the University of Oviedo (Spain). After her PhD, she joined the research group of Prof. Olivier Donard at the IPREM - CNRS (France) until July 2008. Afterwards she joined the University of Oviedo as Research Scientist. She is co-author of more than 80 original research publications and reviews, seven book chapters and one patent. Her research has been presented in more than 100 conferences and has been co-supervisor of six Doctoral Thesis and sixteen Master Thesis Projects. She is principal investigator of several research projects through which her research is funded. Direct correspondence to: fernandezbeatriz@uniovi.es