Spatiotemporal proteomics
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Many diseases are caused by changes in proteins inside the cell. These changes can come from altering the degree to which a gene is expressed by a cell type, making more or fewer copies of a protein. It is increasingly recognized however, that the level of expression of a gene is far from the only way cells control proteins. For instance, proteins can get damaged when they perform their function, and some proteins are more prone to damage. Ensuring that old proteins are effectively recycled over time, i.e., protein turnover, is critical to maintaining cellular activity. In parallel, where inside the cell a protein can be found, i.e., its subcellular location, can have profound influence on its functionality.
Simultaneous Proteome Localization and Turnover Analysis
Researchers have known that these aspects of protein regulation are interlinked. For instance, different compartments within the cell have distinct protein degradation mechanisms responsible for recycling their resident proteins. In the last few years, specialized techniques built on mass spectrometry have allowed scientists to glimpse into these dynamic proteome parameters. However, how to effectively integrate protein turnover measurements with spatial proteomics has remained an unrealized goal. Now in a recently published study by our lab in collaboration of the lab of Dr. Maggie Lam, Associate Professor in Medicine/Cardiology and Biochemistry & Molecular Genetics, we have developed a new method to measure spatial and temporal proteomic changes at the same time.
Mass spectrometry is an important analytical tool in the physical and life sciences, by allowing researchers to measure precisely the identity and quantity of important biological molecules using only minuscule amount of cell and tissue samples. Mass spectrometry is particularly important disease research by directly analyzing proteins on a large scale in what is known as proteomics. The DOM and division cardiology has made investment in mass spectrometry infrastructure, including a recent Strategic Infrastructure for Research Committee (SIRC) grant to establish the Advanced Proteomics Infrastructure (API) directed by our collaborator Dr. Maggie Lam.
To reveal the the dynamics of proteins over space and time, we developed a new experimental approach, which tags proteins with isotopes to record the way they segregate under high speed ultracentrifugation, which allows researchers to deduce where inside the subcellular structures they are found. Using a different type of isotopes, they also measured the rates that heavier elements appear over time in the isolated proteins, as a way to calculate the rate at which cells are recycling proteins. Coupled to mass spectrometry measurements, the results revealed that under cellular stress and cancer drug toxicity, cells changes in protein turnover rates according to which organelles they are found. At the same time, they also found hundreds of proteins that translocate or move from their original sub cellular location to a new locale.
Effect of the cancer drug carfilzomib on cardiac cells
We then applied the method to find out why some commonly prescribed cancer drugs may be toxic to the heart of certain patients. Using human induced pluripotent stem cell (iPSC) models of human heart cells in a dish, we found that surprisingly the cancer drug carfilzomib may disrupt the protein organization in the human heart. On the other hand, as illustrated in the figure, it may specifically block the turnover of certain proteins that work together to generate the contractile force of the heart. This finding may help explain why many patients develop heart failure after cancer drug treatment, and may open up future strategies to reduce the level of toxicity.
By drawing on the power of mass spectrometry and proteomics, this study shows for the first time many novel interactions between protein subcellular localization with protein turnover on a proteome wide scale. Moreover, the method developed can prove to be generally useful for examine protein homeostasis regulations in stress and drug response under many diseases.
This work was supported in part by an NIGMS R35 award to our lab, as well as the generous support of campus funds from the Consortium for Fibrosis Research and Translation (CFReT) directed by Prof. Tim McKinsey and Prof. Mary Weiser-Evans.