The History of MS-Patch-Clamp Technique Development: Integration of Mass Spectrometry and Membrane Electrophysiology

Introduction

In 2021 the journal Nature Methods has published a brilliant paper "Metabolomics profiling of single enlarged lysosomes" [1], in which the authors, in particular, postulate that (quotation): 

• “We therefore built a single-lysosome mass spectrometry (SLMS) platform integrating lysosomal patch-clamp recording and induced nano-electrospray ionization (nano-ESI)/mass spectrometry (MS) that enables concurrent metabolic and electrophysiological profiling of individual enlarged lysosomes”.
• “SLMS can open more avenues for investigating heterogeneous lysosomal metabolic changes during physiological and pathological processes”.

In this outstanding work, the authors have also cited our paper (ref. 22) "MS-Patch-Clamp" or the Possibility of Mass Spectrometry Hybridization with Patch-Clamp Setups for Single Cell Metabolomics and Channelomics" [2] (which is an extended version of our report given in 2014 at the conference “Structure and Functions of Bio-membranes” [3]) in the following context: "In recent years, several reports have suggested and verified the possibility of combining MS with patch-clamp recording for studying single-cell metabolomics (refs. 20-23)”. It can be seen from the latter references that our work is among the pioneering ones in the area of integration of mass spectrometry and patch-clamp, since we are preceded only by the work on the integration of patch clamp electrophysiology and capillary electrophoresis-mass spectrometry [4], while the following works (including the landmark paper on the single-neuron identification of chemical constituents, physiological changes, and metabolism using mass spectrometry published in PNAS [5]) are the works of very productive Chinese authors (partly included in the author team of the paper [1]; see also the Supplement to this article). Indeed, we were the first in the Center for Mass Spectrometry of the Russian Academy of Sciences to propose the MS-patch-clamp concept, which has already emerged in 2010-2011 during the period of our working at the Geochemical Institute of the Russian Academy of Sciences in the laboratory equipped with mass spectrometers, and has been practically tested only in 2014. Subsequently our work has been cited in several papers including those published in Angewandte Chemie [6,7] and also has been used in a number of US and European grant proposals without our participation (e.g., see "Resolving Axonal Clearance using the Ubiquitylation Proteasome Pathway in Alzheimer's Disease (AD)" [8])

The Turning Point

Unfortunately, due to the reorganization of the Research Institutes of the Russian Academy of Sciences after 2013, we have not been able to continue these works for almost ten years. Our instrumentation is extremely outdated (most of the equipment was produced in 1980-2008, such as INCOS MS), so it is impossible to continue this work in Russia now (due to the almost complete absence of the single cell MS equipment). So, we would be happy to collaborate with our international colleagues who have already done everything and even more than we wanted to do about 10 years ago. However, we still have several ideas for the possible improvement and development of the above mentioned methods, and we are ready to share these ideas with them. It is noteworthy that Gradov O.V. tried to continue working on patch-clamp analysis even after the Institute reorganization, but without access to the mass-spectrometric equipment he had to focus on the application of the data analysis methods and other hardware-independent problems, publishing the previously obtained results in the Russian journals usually inaccessible to the international readership, as well as in the conference special issues [9-17]. This forced shift towards theoretical works and reviews resulted from the limited technical basis, leading to the impossibility of working in instrumentally monopolized science in the Russian Federation.

Conclusion

Nevertheless, many well equipped foreign laboratories to date are actively working in this new field and obtain intriguing results in the area of combined single-cell-level mass spectrometry and capillary electrophoresis for the analysis of activity of excitable living tissues [18-29]. Unfortunately, the most remarkable papers in this area are aimed at the single-cell-level proteomics rather than at time-resolved spatiotemporal registration of the ion channel activity and transmembrane transport processes (which were the main aims of our initiative non-profit and non-funded project [30]). Therefore, the most interesting works and technical findings in this field are expected in the nearest future.

References

1. Zhu H, Li Q, Liao T, Yin X, Chen Q, et al. (2021) Metabolomic Profiling of Single Enlarged Lysosomes. Nat Methods 18(7): 788-798.
2. Gradov OV, Gradova MA (2015) "MS-Patch-Clamp" or the Possibility of Mass Spectrometry Hybridization with Patch-Clamp Setups for Single Cell Metabolomics and Channelomics. Advances in Biochemistry 3(6): 66-71.
3. Gradov OV, Gradova MA (2014) On the Possibility of "MS-Patch-Clamp" Or Mass Spectrometry Hybridization with Patch-Clamp Setups for Single Cell Metabolomics and Channelomics. In: Proc. Int. Workshop “Structure and Functions of Biomembranes” (29 Sept. – 3 Oct. 2014. Moscow Institute of Physics and Technology, Russia. 105-106. 
4. Aerts JT, Louis KR, Crandall SR, Govindaiah G, Cox CL, et al. (2014) Patch Clamp Electrophysiology and Capillary Electrophoresis–Mass Spectrometry Metabolomics for Single Cell Characterization. Anal Chem 86(6): 3203-3208.
5. Zhu H, Zou G, Wang N, Zhuang M, Xiong W, et al. (2017) Single-Neuron Identification of Chemical Constituents, Physiological Changes, and Metabolism Using Mass Spectrometry. Proceedings of the National Academy of Sciences 114(10): 2586-2591.
6. Zhang L, Vertes A (2018) Single-Cell Mass Spectrometry Approaches to Explore Cellular Heterogeneity. Angew Chem, Int Ed Engl 57(17): 4466-4477.
7. Zhang L, Vertes A (2018) Einzelzell-Massenspektrometrie zur Untersuchung zellulärer Heterogenität. Angew Chem 130(17): 4554-4566.
8. Tackett AN (2018) Resolving Axonal Clearance using the Ubiquitylation Proteasome Pathway in Alzheimer's Disease (AD).
9. Gradov OV (2015) Multifactor Patch-Clamp Spectroscopy» As A Method for Analysis of the Cell Signalling and Function Regulation via the Ion Channels. Tsitologiya 57(9):625-626
10. Gradov OV (2015) Patch-Clamp-Spectroscopy As A Potential Diagnostic Instrument for Molecular Oncology and Analysis of Ion Channels As A Possible Molecular Targets. Adv Mol Oncol 2(4): 66.
11. Gradov OV (2019) Lock-In Spectral Patch-Clamp Methods and Vesicular Transport Topology from the Standpoint of Structural Stability Concept: Novel Combined Approach based on Digital Signal. Genes & Cells 14(3): 77-78.
12. Gradov OV, Orekhov FK (2016) Correlation Patch-Clamp Spectrometry of Ion Channels – A Combination of Spectral Analysis of the Electrophysiological Response of the Channel in A Non-Strict Time and Methods of Spectroscopy of Ion Channels As Coordination (Complex) Structures. Biomedical Engineering and Electronics 2(13):5-28.
13. Gradov OV, Jablokow AG (2017) Oncoimmunological Aspects of Organellography-Assisted Patch-Clamp-Spectroscopy in Diagnostics. Advances in molecular oncology 4(4):136.
14. Adamovich ED, Alexandrov PL, Gradov OV (2017) Lock-In/Phase-Sensitive Spectral Nanovoltmetric Patch-Clamp with Frequency Discrimination (φ-ω-Patch-Clamp) as Simple Technology for Single Ion Channel Registration in Cellular Biomedicine (Including Cell Channelopathy Diagnostics). European Journal of Medicine. Series B, (4): 30-58.
15. Gradov OV (2020) Mycological Patch-Clamp-Spectroscopy and Patch-Clamp Enzymography: from Saccharomyces Cerevisiae to Patch-Clamp-Assisted Taxonomy Based on Membrane Bioenergetics, Chemiosmotic Coupling and Enzyme Kinetics. Current Mycology in Russia (8):404-406.
16. Adamovich ED, Gradov OV (2023) Planar Patch-Clamp and Patch-Clamp Spectroscopy Methods in (Cardiovascular) Pharmacotherapy. Therapy 9(Special Issue) Cardiovascular Pharmacotherapy.
17. Orekhov FK, Adamovich ED, Gradov OV (2023) Planar Patch-Clamp and Patch-Clamp Spectroscopy Techniques for Antimicrobial Therapies. International conference “Infectious diseases and antimicrobial agents / Xalqaro ilmiy – amaliy konferensiya: “Yuqumli kasalliklar va antimikrob vositalar. Bukhara State Medical Institute, Bukhara, 170-171.  
18. Lombard-Banek C, Choi SB, Nemes P (2019) Single-Cell Proteomics in Complex Tissues Using Microprobe Capillary Electrophoresis Mass Spectrometry. Methods in Enzymology 628: 263-292.
19. Lombard-Banek C, Yu Z, Swiercz AP, Marvar PJ,  Nemes P (2019) A Microanalytical Capillary Electrophoresis Mass Spectrometry Assay for Quantifying Angiotensin Peptides in the Brain. Anal Bioanal Chem 411: 4661-4671.
20. Danser AJ, Poglitsch M (2019) A Microanalytical Capillary Electrophoresis Mass Spectrometry Assay for Quantifying Angiotensin Peptides in the Brain. Anal Bioanal Chem 411(30): 8163-8163.
21. Lombard-Banek C, Yu Z, Swiercz AP, Marvar PJ,  Nemes P (2019) Response to Letter to the Editor regarding “A microanalytical capillary electrophoresis mass spectrometry assay for quantifying angiotensin peptides in the brain”. Analytical and bioanalytical chemistry 411: 8165-8166.
22. Choi SB, Polter AM, Nemes P (2021) Patch-Clamp Proteomics of Single Neuronal Somas in Tissue Using Electrophysiology and Subcellular Capillary Electrophoresis Mass Spectrometry. BioRxiv preprint 2021-09.
23. Choi SB, Polter AM, & Nemes P (2021) Patch-Clamp Proteomics of Single Neurons in Tissue Using Electrophysiology and Subcellular Capillary Electrophoresis Mass Spectrometry. Analytical chemistry 94(3): 1637-1644.
24. Choi SB, Mun?oz-LLancao P, Manzini MC, Nemes P (2021) Data-Dependent Acquisition Ladder for Capillary Electrophoresis Mass Spectrometry-Based Ultrasensitive (Neuro) Proteomics. Anal Chem 93(48): 15964-15972.
25. Nemes P (2021) Mass Spectrometry Comes of Age for Subcellular Organelles. Nat Methods 18(10): 1157-1158.
26. Shen B, Pade LR, Choi SB, Muñoz-LLancao P, Manzini MC, et al. (2022) Capillary Electrophoresis Mass Spectrometry for Scalable Single-Cell Proteomics. Frontiers in Chemistry 10: 863979.
27. Choi SB, Vatan T, Alexander TA, Zhang C, Mitchell SM, et al. (2023) Microanalytical Mass Spectrometry with Super-Resolution Microscopy Reveals A Proteome Transition During Development of the Brain’s Circadian Pacemaker. Anal chem 95(41): 15208.
28. Jia D, Nemes P (2024) Development and Validation of Robocap, A Robotic Capillary Platform to Automate Capillary Electrophoresis Mass Spectrometry En Route to High-Throughput Single-Cell Proteomics. Anal Chem 96(42): 16985-16993.
29. Pade LR, Stepler KE, Portero EP, DeLaney K, Nemes P (2024) Biological Mass Spectrometry Enables Spatiotemporal ‘Omics: from Tissues to Cells to Organelles. Mass Spectrom Rev 43(1): 106-138.
30. Gradov OV (2014-2018) Russian Modulation Patch-Clamp-Spectroscopy Project Initiative Proposal. Gradov Group, Russia.