In this interview conducted at Pittcon 2024, we spoke to Professor John Yates from the Scripps Research Institute about capturing cardiomyocyte cell-to-cell heterogeneity via shotgun top-down proteomics.
Could you please introduce yourself and briefly describe the main focus of your research and what inspired you to start your work in proteomics?
My name is John Yates, and I am at the Scripps Research Institute. For 40 years, my research has focused on protein analysis by mass spectroscopy. I was a zoology major in college, and I loved biology. Through these studies, I developed an interest in sequencing proteins.
A group at my college used mass spectrometry to sequence proteins, and I thought it was a very exciting area of research. Computers were new and fairly rare back then, so it was interesting that a computer was on the mass spectrometer. This experience and exposure to the field inspired me to base my research on utilizing mass spectrometry to sequence proteins.
Your research emphasizes the importance of understanding cell-to-cell heterogeneity. How did you initially identify this as a crucial area of study in the context of health and disease?
To help us understand cell-to-cell heterogeneity, it should be appreciated that cells are different, particularly if you look at them in the context of a functioning tissue. A cell can be considered a system; therefore, tissues are just a system of systems.
The basic aim is to understand cells and their differences, even cells of the same type. These differences often arise as a reaction to their local environments.
The natural evolution from acquiring this basic knowledge is exploring it in the context of health and disease. The ease of studying different aspects of biology has increased since the techniques for carrying out proteomics have become more powerful. Investigations in this field rapidly moved from cells to tissues to whole organisms.
Can you elaborate on the significance of cell-to-cell heterogeneity in understanding biological functions and disease mechanisms?
The effect of protein mutations can vary hugely; in one cell type, a mutation can be devastating, whereas in another protein, the same mutation can be a passenger, not affecting the cell's biology or function.
An example of protein mutations having a devastating effect is in ALS, Lou Gehrig's disease. The mutations that occur in the protein superoxide dismutase destroy motor neurons, and people begin losing muscular function.
What are some challenges and potential advancements in applying top-down proteomics for individual cell analysis?
Proteomics primarily centers around digesting proteins into peptides for analysis. This approach is driven by the fact that peptide analysis technologies are more mature and reliable compared to those for whole proteins.
Proteins pose greater challenges due to their diverse and often extreme chemistry. For instance, membrane proteins are varied, highly hydrophobic, and notoriously difficult to handle, as are proteins such as mucins, which have high levels of glycosylation.
Dealing with intact proteins creates numerous difficulties, amplifying the complexity of analysis. Consequently, top-down proteomics, which focuses on studying intact proteins, lags behind bottom-up proteomics in its capacity to address crucial biological questions.
Image Credit: Proteomics/Shutterstock.com
Despite these drawbacks, there are also benefits associated with using top-down proteomics. For instance, utilizing top-down proteomics is what has driven the field to be able to analyze proteoforms.
Proteoforms are proteins that are modified differently from the same gene. The gene can be spliced to create proteins with differences, including variations in size. These proteins are modified depending on their particular function.
Top-down proteomics is the best form of analysis for multiple investigations. It defines a protein's function and understands how the function can be changed based on the various modifications or patterns of modifications to a protein.
What are the key advantages of employing a CellenONE device and EThcD over traditional methods in your approach using shotgun top-down proteomics?
CellenONE is a new device that is very good at isolating cells. I do not have one in my lab, but I collaborate with a group that has one, and they have figured out how to isolate cardiomyocytes successfully.
The video feed associated with the CellenONE device can be used to observe the cardiomyocytes. It was discovered that they remain beating as they pass through the system, suggesting cardiomyocytes survive the analytical process.
CellenONE has revolutionized the field by discovering a novel way to isolate cells using flow cytometry.
What were some of the biggest technical challenges you faced in developing this method for analyzing single cardiomyocyte cells?
As with many analytical methods, not all issues have been solved yet.
A significant challenge emerged in analyzing the contractile machinery in cardiomyocytes because the proteins responsible for their beating are not very soluble.
Pittcon Thought Leader: John Yates
Therefore, researchers must find a way to make these proteins soluble enough to pass through the mass spectrometer to result in an accurate analysis.
Your study identified a high degree of proteoform heterogeneity among individual cardiomyocyte cells. How does this finding alter our understanding of cardiac biology?
This is a novel approach; therefore, we have not yet gathered enough information for our understanding of cardiac biology to be altered. However, we are on the pathway to doing so. Moving forward, one of our primary objectives is to understand if there is a biological difference between cardiomyocytes of different sizes. Cardiomyocyte size can range from 40 to 100 microns, and these differences may change the function and where it is located in the ventricle heart.
You mention the discovery of numerous post-translational modifications like crotonylation and phosphorylation. Could you tell us more about this finding and what implications these findings have for future cardiac research or therapies?
The important thing to take from these findings is that we can now measure these post-translational modifications. However, it is still too soon to understand the implications for cardiac research and therapies.
Now that these observations have been recognized on normal hearts, we can move forward to start looking at diseased hearts to understand how the phosphorylation patterns may change as a function of disease.
Consequently, we will likely start understanding the implications behind post-translational modifications from these comparisons.
Studies have also been conducted on bulk cells. Through this analysis, it has been observed that changes in phosphorylation patterns could be a function of heart failure. Efforts to understand what drives these changes could help us in the search for new therapies.
What are the next steps in your research? Are there specific areas or applications in cardiomyocyte proteome analysis that you are particularly excited to explore?
An important focus for the future of this field is to gain access to and analyze proteins of a greater size within cardiomyocytes. This will be achieved through continued improvements to the analytical methods used. Alongside this, focus will be placed on gaining more reliable information regarding where the modifications are located in each protein, improving the proteins' fragmentation process.
Overall, the aim is to analyze all the proteins within cardiomyocytes in greater detail, whereas, currently, analysis has only occurred on a fraction of proteins.
As we mark the 75th anniversary of Pittcon, could you share your first memory or experience of attending this conference and how it impacted your view of the scientific community?
Going back 30 years, walking down the exposition hall was a mind-blowing experience. The conference was huge, with 30,000 people and lots of excitement. Many of the vendors were running special events around the conference. Therefore, my overall experience was super interesting and exciting.
At my first Pittcon, I was on the editorial advisory board for the journal, Analytical Chemistry.
About John Yates
John R. Yates is the Ernest W. Hahn Professor in the Departments of Molecular Medicine and Neurobiology at The Scripps Research Institute. He received a B.A. in Zoology and an M.S. in Chemistry from the University of Maine at Orono. He obtained his Ph.D. in Chemistry at the University of Virginia in the laboratory of Donald F. Hunt, with a dissertation titled, "Protein Sequencing by Tandem Mass Spectrometry..He performed postdoctoral research in the laboratory of Leroy E. Hood at California Institute of Technology. At the University of Washington, he obtained the rank of Associate Professor with tenure before moving to The Scripps Research Institute in LaJolla, CA. His research interests include development of integrated methods for tandem mass spectrometry analysis of protein mixtures, bioinformatics using mass spectrometry data, and biological studies involving proteomics. He is the lead inventor of the SEQUEST software for correlating tandem mass spectrometry data to sequences in the database and developer of the shotgun proteomics technique for the analysis of protein mixtures. His laboratory has developed the use of proteomic techniques to analyze protein complexes, posttranslational modifications, organelles and quantitative analysis of protein expression for the discovery of new biology. Many proteomic approaches developed by Yates have become a national and international resource to many investigators in the scientific community. He has received the American Society for Mass Spectrometry research award, the Pehr Edman Award in Protein Chemistry, the American Society for Mass Spectrometry Biemann Medal, the HUPO Distinguished Achievement Award in Proteomics, Herbert Sober Award from the ASBMB, and the Christian Anfinsen Award from The Protein Society, the 2015 ACS’s Analytical Chemistry award, 2015 The Ralph N. Adams Award in Bioanalytical Chemistry, the 2018 Thomson Medal from the International Mass Spectrometry Society, the 2019 John B. Fenn Distinguished Contribution to Mass Spectrometry award from the ASMS, the 2019 HUPO Award in Discovery, and the 2024 Pittsburgh Society Award in Analytical Chemistry. He was ranked by Citation Impact, Science Watch as one of the Top 100 Chemists for the decade, 2000-2010. He was #1 on a List of Most Influential in Analytical Chemistry compiled by The Analytical Scientist 10/30/2013 and is on the List of Most Highly Influential Biomedical Researchers, 1996-2011 (European J. Clinical Investigation 2013, 43, 1339-1365) and the Clarivate List of Highly Cited Scientists in 2015 and 2019-2023. He has published over 1000 scientific articles with >173,000 citations, and an H index of 205 (Google Scholar). Dr. Yates served as an Associate Editor at Analytical Chemistry for 15 years and is currently the Editor in Chief at the Journal of Proteome Research.
About Pittcon
Pittcon is the world’s largest annual premier conference and exposition on laboratory science. Pittcon attracts more than 16,000 attendees from industry, academia and government from over 90 countries worldwide.
Their mission is to sponsor and sustain educational and charitable activities for the advancement and benefit of scientific endeavor.
Pittcon’s target audience is not just “analytical chemists,” but all laboratory scientists — anyone who identifies, quantifies, analyzes or tests the chemical or biological properties of compounds or molecules, or who manages these laboratory scientists.
Having grown beyond its roots in analytical chemistry and spectroscopy, Pittcon has evolved into an event that now also serves a diverse constituency encompassing life sciences, pharmaceutical discovery and QA, food safety, environmental, bioterrorism and cannabis/psychedelics.
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