New Trends in ICP-MS: On the Way from Single Cell to Single Protein Detection

Inductively coupled plasma–mass spectrometry (ICP-MS) is already often applied to single-cell analysis in various applications. For this purpose, a time-gated detection mode with short integration times is used, an approach that is used in the well-established single particle (SP) mode for detection of single metallic nanoparticles (NPs). For detection of single cells, this measuring mode is given the name single cell (SC) mode (see more details at [1,2]). This relatively new method is used directly to detect toxic or essential metals and hetero-elements in biomolecules, metal-containing pharmaceuticals, or NPs in single cells. With conventional ICP-MS instruments, the number of elements that can be detected is limited because of the short integration time, of a few milliseconds, so that mainly just a single isotope can be detected. Therefore, for multielement analysis of single cells at short integration times, time-of-flight (TOF) mass spectrometers are better suited for detection and the first examples have been published (3). In contrast to direct metal detection, however, the detection of metals in metal-tagged antibodies is even more frequently applied. Historically, the state-of-the-art method for characterizing proteins (biomarkers) or cell receptors of many cells individually has been flow cytometry with fluorescence detection (4). But if multiple parameters must be analyzed simultaneously, this optical method is limited in the number of applicable fluorescence colors by spectral overlap, fluorescence dye quenching, and autofluorescence of the sample itself. Therefore, a novel high-throughput technique for real-time analysis of multi-parameter assays of single cells based on ICP-TOF-MS (known as CyTOF) was developed by Scott D. Tanner and colleagues (and is now commercially available by the company Fluidigm), which is known as mass cytometry (MC) (5). In this method, antibodies are bioconjugated with metal tags (for more details about the history, chemistry, and applications of mass cytometry, see [6–8]) and are applied to detection of cells suspended in a buffer solution. The cell suspension is introduced into the CyTOF-MS by a pneumatic nebulizer. More recently, a laser ablation (LA) system was coupled to CyTOF-MS devices and this new application was given the name imaging mass cytometry (more details are given in [9]) although it is nothing other than conventional LA-ICP-MS but with cellular lateral resolution. 

For metal tagging of antibodies polymer tags have been developed carrying chelating compounds that can be loaded with enriched isotopes of the rare earth elements (REE) (10). These elements are exclusively applied in mass cytometry because of their similar chemistry and presently up to 40 single and enriched isotopes can be used in a single assay, so that a multiparametric analysis is possible. The sensitivity of the assay is directly proportional to the number of atoms covalently attached to the antibody, resulting in a signal amplification for each antibody by a factor from 20 to 100. Given the fact that each single target cell might have thousands of receptors (proteins embedded in a cell membrane) on the cell surface to which these antibodies can bind, an additional signal amplification for each cell can be expected, so that in total currently the amplification factor is in the range of 10⁵ and this allows detection of the most abundant biomarkers.

However, the number of proteins in a cell ranges from 10⁶ down to about a hundred for less-abundant proteins. For the detection of these less-abundant proteins in a single cell assay, much higher degrees of tagging are required. Metallic nanoparticles (with diameters of around 50 nm) contain many millions of atoms and have been used for tagging antibodies successfully and were used successfully, for instance, in secondary electron microscopy. However, the application of this approach in immunoassays for ICP-MS detection is still advancing only slowly, although a gain proportional to the number of atoms in the nanoparticle looks very promising. If such a gain were realized for mass spectrometry, then this will definitely cause another revolution in all bio-related sciences very similar to the quantum jump initiated by the polymerase chain reaction (PCR) with real-time PCR, which excels by an amplification factor of more than a million for DNA detection (11). Such a gain would be desirable for fundamental quantitative proteomics as well as for detection of novel, less-abundant biomarkers (proteins) for many different diseases and would enable easy personalized diagnosis at tolerable costs. 

A first example of this was published by Cruz-Alonso and associates, who studied the function of metallothionein (MT) during oxidative stress in the human eye (12). They used primary antibodies directed against MT 1/2 and MT 3 which were tagged by Au clusters containing more than 500 atoms of Au. This tagging resulted in a significant signal improvement, such that MTs could be detected even in different layers of tissue with high lateral resolution. In the work of Drescher and associates, the authors were able to detect single Au and Ag NPs in single fibroblast cells using LA-ICP-MS for single cell imaging (13). Therefore, Tvrdonova and associates used antibodies bioconjugated with gold nanoparticles (AuNPs) with diameters from 10 to 60 nm for detection of immunoglobulins by LA-ICP-MS (14). Dot blot experiments were performed to demonstrate that the binding capability of the antibody was not compromised by such a large particle.

So far, both examples show that tagging of antibodies by NPs is in general possible without compromising the binding capability of the antibody, but single metal NPs do not allow multiparametric detection in mass cytometry. However, reproducible production of NPs directly from REEs is quite challenging. Therefore, Scharlach and associates used very small iron oxide nanoparticles (VSOP) with a diameter of only 6.6 nm (measured by transmission electron microscopy) that is synthesized by a precipitation reaction (15). They added europium, an element of the REE group, to the precipitation solution and ~30 europium atoms per particle were doped into the VSOP bulk containing 4500 iron atoms. Because of the similar chemistry of all REEs, even larger NPs can be synthesized using modified reaction conditions and alternative REE elements soluble in the reaction solution can be incorporated so that the number of distinguishable tags can be increased significantly, to enable multiplex or multiparametric assays in mass cytometry.

The same idea was followed by Grunert and associates, who developed a simple solvothermal synthesis of Eu³⁺-doped gadolinium orthovanadate nanocrystals (Eu:GdVO4) that already contained two different lanthanides, for multimodal applications (16). The Eu³⁺ doping of the vanadate matrix provides red photoluminescence after illumination with ultraviolet (UV) light so that NPs in cells can be localized by optical microscopy. Gd³⁺ ions in the nanocrystals reduce the T1 relaxation time of surrounding water protons at the surface of the crystals, allowing these nanocrystals to act as a positive magnetic resonance imaging (MRI) contrast agent as well. Synthesis resulted in polycrystalline nanocrystals with a crystal size of 36.7 nm. The nontoxic nanocrystals were subsequently used for intracellular labeling of both human adipose-derived stem cells and adenocarcinomas human alveolar basal epithelial (A549) cells and imaged in the cells by LA-ICP-MS, MRI, and fluorescence detection. Thus,  these crystals look very promising for application in mass cytometry and multimodal spectroscopies.

Pichaandi and coworkers used a similar idea to develop REE-doped NPs for the tagging antibodies in mass cytometry (17). They examined the use of silica-coated NaHoF₄ NPs (with an ~12 nm core diameter and a 5 nm silica shell) as a model for coprecipitation of REE in a host matrix. They compared the sensitivity of NP–antibody conjugates to polymer–antibody conjugates toward seven biomarkers with varying expression levels across six different cell lines; a very promising 30 to 450-fold signal enhancement was seen for the NP-based reagent, which contained only 12.000 Ho atoms per NP.

A very new signal amplification strategy was also demonstrated by Yuan and associates (18). They constructed an element-tagged virus-like nanoparticle (VLNP) with a precise number of atoms. The VLNP was applied as a membrane-specific cell biomarker that provided additional amplification because many VLNPs were able to bind to the cell membrane, which resulted in significantly higher sensitivity in a cell counting system using ICP-MS. With this approach, signal amplification of more than two orders of magnitude was achieved. In this example a VLNP was used as a metal-tagged probe, but in principle detection of viruses (such as COVID-19) itself could become possible if adequate and specific antibodies are available. 

All the examples mentioned are proof-of-principle experiments only; in the future, it must be demonstrated that they can be applied in complex biological systems. Nevertheless, the application of NPs for metal-tagging looks promising for detection of less-abundant proteins, and this author is convinced that in the very near future, routine single-protein detection of even less abundant biomarkers by ICP-MS and CyTOF-technologies will become possible, revolutionizing analytical proteomics and medical diagnosis.

We never thought, during the past 35 years of reading this journal, that inorganic mass spectrometry (ICP-MS) would become an important analytical method for medical research and diagnosis to improve, ensure and preserve our future health! Stay healthy! 

References

1.    L. Mueller, H. Traub, N. Jakubowski, D. Drescher, V. I Baranov, and J. Kneipp, Anal. Chem. 10, 6963–6977 (2014).

2.    H. Wang, M. He, B. Chen, and B. Hu, J. Anal. At. Spectrom. 32, 1650–1659 (2017).

3.    K. Löhr, O. Borovinskaya, G. Tourniaire, U. Panne, and N. Jakubowski, Anal. Chem. 91, 11520–11528 (2019).

4.    J. Picot, C.L. Guerin, C. Le Van Kim, and C.M. Boulanger, Cytotechnology 64, 109–130 (2012).

5.    S.D. Tanner, D. Bandura, O. Ornatsky, V. Baranov, M. Nitz, and M.A. Winnik, Pure Appl. Chem. 80, 2627–2641 (2008).

6.    M.H. Spitzer and G.P. Nolan, Cell 165, 780–791 (2016).

7.    C. Giesen, L.Waentig, U. Panne, and N. Jakubowski, Spectrochim. Acta, Part B 76, 27–39 (2012).

8.    G. Schwarz, L. Müller, S. Beck, and M. W. Linscheid, J. Anal. At. Spectrom. 29, 221–233 (2014).

9.     Q. Chang, O. I. Ornatsky, I. Siddiqui, A. Loboda, V.I. Baranov, and D.W. Hedley, Cytometry Part A 91, 160–169 (2017).

10. X. D. Lou, G. H. Zhang, I. Herrera, R. Kinach, O. Ornatsky, V. Baranov, M. Nitz, and M. A. Winnik, Angew. Chem. Int. Ed. 46, 6111–6114 (2007).

11. R. Saiki, S. Scharf, F. Faloona, K. Mullis, G. Horn, H. Erlich, and N. Arnheim, Science 230, 1350–1354 (1985).

12. M. Cruz-Alonso, B. Fernandez, L. Álvarez, H. González-Iglesias, H. Traub, N. Jakubowski, and R. Pereiro, Microchimica Acta 185, 64–72 (2017).

13. D. Drescher, C. Giesen, H. Traub, U. Panne, J. Kneipp, and N. Jakubowski, Anal. Chem. 84, 9684–9688 (2012).

14. M. Tvrdonova, M. Vlcnovska, L. Pompeiano Vanickova, V. Kanicky, V. Adam, L. Ascher, N. Jakubowski, M. Vaculovicova, and T. Vaculovic, Anal. Bioanal. Chem. 411, 1–6 (2018).

15. C. Scharlach, L. Mueller, S. Wagner, Y. Kobayashi, H. Kratz, M. Ebert, N. Jakubowski, and E. Schellenberger, J. Biomed. Nanotech. 12, 1001–1010 (2016).

16. B. Grunert, J. Saatz, K. Hoffmann, F. Appler, D. Lubjuhn, N. Jakubowski, U. Resch-Genger, F. Emmerling, and A. Briel, ACS Biomater. Sci. Eng. 4, 3578–3587 (2018).

17. J. Pichaandi, G. Zhao, A. Bouzekri, E. Lu, O. Ornatsky, V. Baranov, M. Nitz, and M.A. Winnik, Chem. Science 10, 2965–2974 (2019).

18. R. Yuan, F. Ge, Y. Liang, Y. Zhou, L. Yang, and Q. Wang, Anal. Chem. 91, 4948–4952 (2019).

Norbert Jakubowski is the former Head of Division at the German Federal Institute for Materials Research and Testing (BAM) and is currently a consultant with Spetec GmbH, in Erding, Germany. Direct correspondence to norbi.jakubowski@gmail.com