Single-Cell Proteomics: Mass Spec vs Single-Molecule Sequencing

Unlike bulk proteomics, which analyzes average protein expression across a group of cells, single-cell proteomics explores the heterogeneity and diversity of single cells. These deeper insights offer a new understanding of cell biology, cellular responses to stimuli and how complex signaling pathways contribute to the cell’s function as a system. When studying human health and disease, this field is set to have a profound impact on how we diagnose and treat illnesses in personalized medicine. Medical fields such as cancer research are already benefitting from single-cell proteomics, which can shed light on why certain cells within a tumor respond to a specific therapy, while others may not, for example.

The single-cell proteomics toolbox continues to evolve, with novel workflows combining a variety of analytical tools being published frequently. The dominant set-up for most experiments involves the use of mass spectrometry (MS) in some configuration. Lately, there has been excitement surrounding single-molecule sequencing and its possible applications in proteomics.

Dr. Ryan Kelly is an associate professor in the Department of Chemistry and Biochemistry at Brigham Young University. His research centers around the development of new technological solutions for ultrasensitive biochemical analyses, including single-cell profiling and high-resolution proteome imaging. Technology Networks interviewed Kelly to hear his thoughts on the single-cell proteomics landscape and the potential impact of single-molecule analysis. 

Molly Campbell (MC): For readers that are unfamiliar, how does single-cell MS proteomics differ from single-molecule protein sequencing?

Ryan Kelly (RK): Thousands of copies of a given peptide are needed to make a confident measurement by MS, and this is what we do with single-cell proteomics. The non-MS–based single-molecule protein sequencing approaches (based on nanopores, etc.) measure individual copies of each peptide, but these have not been used to measure peptides or proteins in single cells yet. The fields of single-cell proteomics and single-molecule protein sequencing will converge at some point, but they haven’t yet.

MC: Many MS vendors are creating instruments that support single-cell MS proteomics. Why would single-cell protein sequencing be advantageous, given the advancements in the MS space? Could this be adopted in complement to single-cell MS proteomics, or do you envision this could be a technology that competes with MS?

RK: Single-cell MS is more “ready for prime time”, while single-molecule sequencing is generally still in a proof-of-concept stage. I suspect that as single-molecule sequencing matures, there will be areas of complementarity and areas of competition. For example, single-molecule sequencing technologies by their very nature detect single molecules, while MS requires a few thousand or tens of thousands of copies to make a measurement. So sequencing approaches could potentially fill in the gap on ultra-trace–level proteins, particularly if those low-abundance species can be separated from the higher abundance ones.

MC: What are the key challenges faced by both single-cell MS proteomics and protein sequencing?

RK: Both technologies have dynamic range and measurement throughput limitations. More effective sample preparation and separations can also benefit both fields.

MC: You have previously said that you believe “rumors of mass spectrometry’s demise have been greatly exaggerated”. Can you expand on this viewpoint?

RK: The non-MS proteomics technologies tend to be purpose-built for a given application or have a predefined panel of target analytes. If you want to study something outside of that range, you’re out of luck. In contrast, the same mass spectrometer can be used to study protein digests, intact proteins, post-translational modifications, lipids, carbohydrates, etc., from all biological systems. It’s this versatility that will allow MS to fill in the gaps even as other emerging technologies mature.

MC: How do you see the proteomics research field evolving over the coming years?

RK: If single-cell proteomics continues to advance to where it becomes very high throughput and very sensitive, it could become the default mode of proteome profiling. A similar trend is already happening with transcriptomics. Measuring populations of cells one at a time provides both that single-cell granularity and population-level information at the same time.  

Dr. Ryan Kelly was speaking to Molly Campbell, Senior Science Writer for Technology Networks.