Abstract 15624: Detailed Proteomic Analysis Of Mouse And Human Cardiac Membrane Proteins Reveals Novel Cardiac Proteins
Introduction: Membrane proteins are involved in a variety of key biological functions; however they are a challenging class of proteins to investigate, largely due to their relative low abundance and their hydrophobic nature. Recent advances in mass spectrometry-based proteomics have enabled the large-scale analysis of this vital cell compartment. We carried out a large scale proteomic study of mouse and human cardiomyocytes, smooth muscle cells and endothelial cells in which we identified unique proteins for all three cardiac cell types with a primary focus on unveiling novel proteins essential to cardiac function.
Methods and Results: We used cationic silica bead-coating to enrich for cell-surface associated proteins from primary mouse neonatal, human fetal cardiomyocytes, as well as human endothelial and smooth muscle cells. Shot-gun proteomics identified >3,000 mouse proteins and >2,500 human proteins. Using QSpec statistical analysis we calculated differential spectral counts between proteins found in the membrane enriched and membrane depleted fraction and provide a dataset of 555 cardiomyocytes proteins which include many known membrane proteins. Bioinformatic integration with transmembrane helix predictions, Phenotype Ontology (PO), and publically available microarray data sets, identified a rank ordered set of cardiac-enriched surface proteins; select examples of which the subcellular location were further confirmed using high resolution confocal microscopy, immunogold electron microscopy, and sucrose density gradient biochemistry. For several of the highly ranked membrane proteins, lentiviral-based shRNA knock-down demonstrated significantly altered calcium transient amplitudes, calcium release rates, and calcium uptake rates in both neonatal mouse cardiomyocytes and in human embryonic stem cell derived cardiomyocytes.
Conclusion: In conclusion, we have provided the first comprehensive analysis of membrane cell surface-associated proteins in all three major cardiac cell types, and provided a pipeline to validate surface proteins in cardiomyocytes that might be critically involved in calcium dynamics.
- © 2011 by American Heart Association, Inc.