Single cell technologies are changing our understanding of human health and disease. Single cell genomics and transcriptomics are mature disciplines that can study more than a million single cells (1). However, human
Biology cannot be understood by analyzing DNA and RNA alone. It also requires the study of proteins and protein modifications, lipids and metabolites.
Proteins are biochemically active and also serve as signaling molecules. So it’s not surprising that about 95% of drugs target proteins. However, protein molecules cannot be amplified like DNA or RNA to perform single-cell proteomics measurements. Therefore, novel and highly sensitive technologies are required that can unravel this complexity at the single-cell level and contribute to our understanding of emerging problems related to health and disease (2).
Single cell omics landscape
Protein function is often modulated by post-translational modifications such as phosphorylation and ubiquitination, which can alter the course of cell function with rapid kinetics. Processes such as endogenous proteolysis and glycosylation are known to play a role in oncological mechanisms (3,4). In addition, gene expression is influenced by so-called bursts in expression, which leads to additional variations that would be automatically normalized in proteins by post-translational regulation processes (5). In addition, alternative splicing of RNA transcripts can result in additional protein variants. Thanks to the pioneering work of a relatively small group of dedicated scientists and the advent of extremely sensitive mass spectrometers, single-cell proteomics technologies are now entering the mainstream. Typical estimates of the protein content of individual cells are on the order of 200 picograms (that’s one billionth of a milligram). In a recent study, qualitative and quantitative information was obtained for up to 1400 proteins from single cells using an unbiased, single-proteomics approach that did not require complex isobaric labeling chemistries to amplify peptide signals (6). Cluster analysis of the data could distinguish cell types and cell cycle stages, although the technology does not specifically target known and verified markers.
This microheterogeneity in seemingly homogeneous cell populations plays a key role in critical signaling pathways in biological systems. The underlying microheterogeneity is caused by variations in genes and their expression, and understanding these variations at the single-cell level helps identify the few cells that serve, for example, as the seed for carcinogenesis. The study of DNA and RNA molecules within the cell is one of the most common approaches in single-cell biology and has also helped motivate the measurement of proteins at the single-cell level. Exponential advances have been made in single-cell DNA and RNA sequencing technologies, and depending on the application, a variety of sequencing techniques can be used for these studies (2).
With the help of such advanced technologies, studies involving the measurement of single-cell transcriptomes from more than a million single cells are now possible (1) and have revealed previously unseen biology and highlighted the heterogeneity of single cells – opening up new areas of biology and Medicine. A common factor in all of these approaches is the ability to amplify DNA and RNA molecules to virtually any desired level, bringing those molecules into a detectable or quantifiable range (7).
Unbiased single-cell proteomics have been performed in recent years by specialized research groups working on nanofluidics, which has not yet been readily adopted by the general research community. These applications often focus on minimizing loss during sample preparation and multiplexing samples to increase signal intensity (8,9). Despite these solutions, however, there is still a need for innovations that can increase the sensitivity of the mass spectrometer.
Captured ion mobility spectrometry
The development of parallel accumulation and serial fragmentation (PASEF) (10) provided a spectroscopic technique that was used with liquid chromatography in conjunction with mass spectrometric (LC-MS)-based proteomics to improve sequencing speed and sensitivity. PASEF makes efficient use of the ion beam and together with the intelligent precursor selection of ions eluting from a TIMS (Trapped Ion Mobility Spectrometry) cycle, achieves fast MS/MS identification speed. In addition, the ions within the TIMS cell are spatially and temporally focused, which leads to a significant increase in sensitivity. This enables the analysis of small sample amounts in the range of low nanogram peptide loadings.
TIMS measurements also provide collision cross-section (CCS) values and separation of isomeric species that are mobility-displaced but mass-aligned, and reduce ratio compression in multiplex quantification approaches. The introduction of these 4D proteomics capabilities has bridged the gap between the needs of the most demanding proteomics approaches – such as clinical research proteomics, companion diagnostic research and personalized medicine research – and the solutions effectively available on the market.
Next-generation sequencing technologies now represent a multibillion-dollar industry (11) that promises to help deliver personalized medicine and precision therapeutics that will help combat complex and heterogeneous diseases such as cancer and Alzheimer’s.
Single-cell protein technologies have the potential to change our understanding of cell biology at the macromolecular level and answer fundamental questions about protein dynamics, cell differentiation trajectories and disease mechanisms. These processes act at the nano- and microscopic level, but fundamentally influence the higher-order macroscopic behavior. It is therefore important that these processes are understood with the highest possible spatial resolution.
1. J.Cao, et al., Nature 566, 496-502 (2019).
2. G Chen, B Ning and T Shi, Front. geneticsApril 5, 2019.
3. LA Liotta and EF Petricoin, J Clin Invest. 116(1) 26-30 (2006).
4. MA Connelly, et al., J Transl. medication 15, 219 (2017).
5. GK Marinov, et al., genome res. 24, 496-510 (2014).
6. AD Brunner, et al., bioRxiv 12.22.423933 (2020).
7. CFA de Bourcy, et al., Plus one 9 (8) e105585 (2014).
8. D. Hartlmay, et al., bioRxiv 04.14.439828 (2021).
9. N. Slavov, Current opinion in chemical biology 60, 1-9 (2021).
10. F. Meier, et al., Mol Cell Proteomics. 17(12) 2534-2545 (2018).
11. Markets and Markets, Single Cell Analysis Market by Cell Type (Human, Animal, Microbial), Product (Consumables, Instrument), Technique (Flow Cytometry, NGS, PCR, Microscopy, MS), Application (Research, Medical), End User (Pharma, Biotech, Hospitals) – Global Forecast until 2026, marketsundmarkets.comFebruary 2020.
About the author
Gary Kruppa, PhD, is Vice President of Proteomics, Bruker Daltonics.
Volume 46, number 7
When referring to this article, please cite it as G. Kruppa, “Innovations in single-cell proteomics drive advances in disease research,” pharmaceutical technology 46(7) 20-22 (2022).