The rapid development of high accuracy and high sensitivity mass spectrometry technologies has revolutionized our understanding of proteins and their contribution to cellular function. However, there is an increasingly apparent disconnect between these state-of-the-art tools and their effective applications to advance cardiovascular biology and medicine. Despite progress made in certain areas of investigation, cardiovascular proteomic research faces three major challenges: lack of study designs to accurately pinpoint connections between function and proteomes; limited quantitative information to understand the dynamics of proteomes in disease phenotypes; and the overwhelming quantity and fragmented nature of mass spectra datasets lacking functional annotations. This 2012 Thomas Smith Lecture presentation will provide an overview of the accomplishments in cardiovascular proteomics, highlight the barriers and opportunities in this field, and address the aforementioned obstacles using examples from organellar proteomics investigations, whereby model systems and computational tools are combined to understand human diseases.