Feature: Toward Normalization of Quantitative Single Cell ICP-MS Experiments

Because cells are heterogeneous materials appearing in many sizes and containing many different elements depending on the model, inductively coupled plasma–mass spectrometry (ICP-MS) is often used to conduct element analysis of single cells. Maria Montes-Bayón of the Faculty of Chemistry at the University of Oviedo (Asturias, Spain) has been working with single cell ICP-MS (sc-ICP-MS) to study the uptake and apoptotic status of nanoplatinum (IV) treated cells, specifically selenized yeast. Montes-Bayón recently spoke to Spectroscopy about this work.

What makes ICP-MS an effective technique in your experiments for single cell analysis?

Single cell -omics is a growing field of research, since cellular heterogeneity is what triggers the beginning of many diseases. Among single cell -omics techniques (such as genomics, trancriptomics, and proteomics), metallomics is also playing an important role, since metals are key regulators of cell functions. In addition, the monitoring of the differential uptake of drugs containing metals by individual cells might explain their effectiveness. The evaluation of the intracellular content of different elements in individual cells can be performed by ICP-MS using the previously established concept of single particle ICP-MS, considering some critical points (a cell is more sensitive to the analysis process than a particle). Through the years, we have been evaluating the suitability of different ICP-MS based strategies for single cell analysis and I would say we have found the most successful combination for the sensitive and sequential determination (with triple quadrupole-based mass analyzers) of single elements in single cells.

You’ve recently worked with selenized yeast as a reference standard. Why did you choose this standard, and what are the challenges involved in using these cells as a reference standard?

Different approaches are available for individual introduction into ICP-MS. To address the suitability of every system, different authors use different strategies. Reference materials are generally used to “validate” analytical results, but it is uncommon to have reference standards specifically for “cell-based” materials. We realized that selenium enriched yeast certified reference material, or SELM-1, was a yeast material that was certified in total Se content. While talking to microbiologists at our university, they noted that yeast cells were robust enough to be intact over time, and if preserved in the correct way, they should be alive. Thus, we started a multiple-techniques study on the suitability of the reference material (for example, using confocal microcopy of labeled cells, flow cytometry, and ICP-MS analysis). We also found it necessary to address the differential Se content intra- and extracellularly.

Are there any specific sample preparation and analysis conditions that are required to be successful in this analysis?

Indeed, we realized the importance of matrix effects when using ICP-MS. It seems strange that often something so well-known for so many years is not considered carefully when conducting this type of analysis. The paper we published establishes that the reference material can be used as such for sc-ICP-MS if carefully washed a few times and then dried before analysis. If this procedure is not followed, the accuracy of the results might be compromised when using external calibration.

Are there any sample cell types that are easier or more difficult to analyze, whether you learned this through testing or presume it from past experiences?

Yes. In our experience (our first paper is from 2017), we have researched yeast cells (Saccharomyces), bacterial cells (Streptomyces, Salmonella, E. coli),and human cells (A2780, A2780cis, OV-CAR3, A549, MCF-7, MDA-MB 231, STR-747, HepG2 corresponding to ovarian, lung, breast, and liver cancer cells). The first two models (yeast and bacteria) are much more resistant to the nebulization and transport into the plasma so the chance for leaking of the intracellular content into the sample matrix is minimized. With human models, it is much more challenging due to the delicate composition of the cell membrane that can be easily disrupted (3). In some cases, we fix the cells to minimize disruption, but this can be risky if constitutive elements need to be analyzed.

How does this work differ from what has been previously done by yourself or others?

This work tries to address the possible use of an already commercialized reference material to validate single cell ICP-MS analysis data. I think no one has used it for this purpose before.

Were there any limitations or challenges you encountered in your work?

Not really. We just could not believe the results we were obtaining on the unwashed samples, and we thought we were doing something wrong. We had a lot of brainstorming with the two post-docs in my office trying to understand why the results were 30% lower than expected. We even asked a more experienced post-doc to run the same sample and some PhD students to measure the same cell suspension every day to address transport efficiencies. It turns out that it was due to some matrix effects! It took us a while to understand this.

Since the previous interview, Montes-Bayón was awarded the Lester W. Strock Award, given by the New England Section of the Society for Applied Spectroscopy (SAS) to recognize, as noted on the SAS website, “a selected publication of substantive research in/or application of analytical atomic spectrochemistry in the fields of earth science, life sciences, or stellar and cosmic sciences.” This second interview, where we revisit our previous conversation as well as discuss her career in general, is part of an ongoing series of interviews with the winners of awards that are presented at the annual SciX conference, which will be held this year from October 8 through October 13, in Sparks, Nevada.

Since our last talk regarding your work with ICP-MS normalized quantitative experiments using certified selenized yeast, has there been any additional feedback or next steps that you’d like to share?

Well, we never stop! We have moved some steps forward into a different topic in my group which is the use of nanoparticles to transport cisplatin into tumor cells. Part of the work is still unpublished, but we have developed some nice strategies to address their incorporation in 3D cell cultures, which is the natural evolution from 2D cultures and closer to tumor tissues. However, for this we need a strong collaboration from colleagues from biochemistry and biotechnology to create these complex cellular structures from different cell types. We are really excited about the outcome of this project as well.

What does your being named the recipient of the Strock Award mean to you professionally? Personally?

In my case, the award is given to the contributions in speciation and metallomics, basically due to the application of atomic spectroscopy on the field of biomedicine and life science. This makes me particularly pleased since I have been trying to establish collaborative research in these fields for a long time with colleagues from different disciplines. I deeply believe that analytical chemists are well prepared to conduct multidisciplinary research and can provide interesting methodological alternatives in the field of life sciences. Therefore, the prize recognizes the effort required to make this feasible. From a personal point of view, it is particularly important to me since part of my work was conducted in Cincinnati, in the group of Prof. Joe Caruso, and this period completely changed my vision of science. I can be only grateful for those times, from both a personal and a professional perspective.

When did you first determine that a career in science was the path for you?

I think this is something you do not really plan. When I finished my bachelor’s degree, the possibility to go to the UK to do my master’s degree thesis became feasible. Learning English was the first driving force to go there, but I discovered a new passion in research (working with Prof. Hywel Evans, once I could understand his accent from Wales). Then, back to Oviedo, Prof. Alfredo Sanz-Medel offered me the possibility to earn my doctorate in his group, and I further developed my enthusiasm for research.

As you mentioned, you first studied chemistry at the University of Oviedo, then joined the University of Plymouth (UK) in the framework of the Erasmus program for undergraduate thesis studies. You were then in Caruso’s research group in the Department of Chemistry at the University of Cincinnati, Ohio (USA) as a Fulbright postdoctoral fellow from April 2000 to July 2002. What challenges did you encounter in these transitions across multiple departments?

In addition, I was a visiting scientist in Germany for two different periods in Hamburg and Berlin (for a total of seven months). All in all, the different working environments comprise one of the aspects that enriches the scientific life the most. It always requires some time to learn how things are done in the different places but, finally, you gain an open vision about research, and life in general. Being in the United States, where I spent the longest time abroad, gave me the opportunity to learn how to be more independent in research, and how to communicate science more effectively. From all the places I have visited, I have taken something that helped me in the next step of my career.

Tell me a little bit about your first published article.

My first paper dealt with research based on plasmas but microwave- induced plasma, MIP. Soon after, the first ICP-MS arrived in Oviedo, and since I had gained some experience during my time in Plymouth, Prof. Sanz-Medel (one of my doctorate supervisors) reoriented my doctorate to ICP-MS that aimed to develop first a semiquantitative analysis method for the fast analysis of environmental samples (4). Thus, thanks to the time in UK, my doctoral experiments focused on the use of ICP-MS for semi-quantitative total elemental analysis and speciation, which resulted in very successful and productive work.

What do you consider your greatest strength, and how does that strength factor into your professional life?

It is hard to define these things about yourself. I think I am very enthusiastic about the research work we do in the laboratory, and this helps me to communicate it in a clear and somehow entertaining way. Such enthusiasm always helps when writing manuscripts or research projects to get funding. This is very important for both personal and professional aspects.

What do you consider to be your greatest professional accomplishment?

Maybe to keep an independent research group that is growing and attracts the interest of new PhD students, not only from the field of chemistry but also biotechnology or biochemistry.

What advice can you offer those starting out in their post-doctorate career?

I think it is summarized along this interview: One must develop capabilities to adapt to new working environments and to extract the best of each one of them; have enthusiasm about your work; and, while imagination is always welcome, one should be able to listen and understand what colleagues from other fields need.

References

1. Pereira, J. S. F.; Álvarez-Fernández García, R.; Corte-Rodríguez, M.; Manteca, A.; Bettmer, J.; LeBlanc, K. L.; Mester, Z.; Montes-Bayón, M. Towards Single Cell ICP-MS Normalized Quantitative Experiments Using Certified Selenized Yeast. Talanta 2023, 252, 123786. DOI: 10.1016/j.talanta.2022.123786
2. Corte Rodriguez, M.; Álvarez-Fernández García, R.; Blanco, E.; Bettmer, J.; Montes-Bayón, M. Quantitative Evaluation of Cisplatin Uptake in Sensitive and Resistant Individual Cells by Single-Cell ICP-MS (SC-ICP-MS)Anal. Chem. 2017, 89 (21), 11491–11497 DOI: 10.1021/acs.analchem.7b02746
3. Corte-Rodríguez, M.; Álvarez-Fernández, R.; García-Cancela, P.; Montes-Bayón, M.; Bettmer, J. Single Cell ICP-MS Using Online Sample Introduction Systems: Current Developments and Remaining Challenges. TrAC, Trends Anal. Chem. 2020, 132, 116042. DOI: 10.1016/j.trac.2020.116042
4. Montes-Bayon, M.; Garcia Alonso, J. I.; Medel, A. S. Enhanced Semiquantitative Multi-Analysis of Trace Elements in Environmental Samples Using Inductively Coupled Plasma Mass Spectrometry. J. Anal. At. Spectrom. 1998, 13, 277–282. DOI: 10.1039/A707496D

Maria Montes-Bayón is Full Professor in Analytical Chemistry in the Department of Physical and Analytical Chemistry at the University of Oviedo (Spain) since December 2017. She holds a bachelor’s degree in chemistry (1993, University of Oviedo, Spain), a bachelor-thesis in Analytical Chemistry (1994, University of Plymouth, UK and Oviedo), and a PhD (1999, University of Oviedo; awarded) degree. In the year 2000, she joined the research group of Prof. Joseph A. Caruso at the Department of Chemistry at the University of Cincinnati as a Postdoctoral Fulbright Fellow. She went back to Oviedo on a Senior Researcher contract and was promoted in 2008 to Associate Professor, and in 2017 was promoted to Full Professor of Analytical Chemistry. Her main research interests are: a) Development of quantitative strategies for determination of molecular biomarkers of clinical relevance at the single cell level using ICP-MS; b) Evaluation of encapsulated metallodrugs for their potential use in chemotherapy using mass spectrometry: a focus on cellular resistance mechanisms; and c) analytical tools to study biomedical aspects of nanoparticles: biogenic nanoparticles and metal nano-debris from metallic implants. She has supervised 14 PhD students since 2007 and mentor of 5 postdocs who built up a successful career afterwards. She has given >40 invited and plenary lectures in national and international congresses and conferences and co-authors 164 publications (80% in Q1; 32 with >50 citations), including original research articles, reviews, and several book chapters (with >5,300 total citations, H-index = 43). She has got funding through 29 international/national/regional research projects (12 of them as PI) and is associate editor of the journal Metallomics (RSC/OUP) since 2015. Currently, she has been elected as President of Spanish Society for Analytical Chemistry since 2022.