AbstractAtherosclerotic plaque rupture leading to myocardial infarction is a major global health burden. Applying the tandem stenosis (TS) mouse model, which distinctively exhibits the characteristics of human plaque instability/rupture, we use quantitative proteomics to understand and directly compare unstable and stable atherosclerosis. Our data highlight the disparate natures and define unique protein signatures of unstable and stable atherosclerosis. Key proteins and pathway networks are identified such as the innate immune system, and neutrophil degranulation. The latter includes calprotectin S100A8/A9, which we validate in mouse and human unstable plaques, and we demonstrate the plaque-stabilizing effects of its inhibition. Overall, we provide critical insights into the unique proteomic landscape of unstable atherosclerosis (as distinct from stable atherosclerosis and vascular tissue). We further establish the TS model as a reliable preclinical tool for the discovery and testing of plaque-stabilizing drugs. Finally, we provide a knowledge resource defining unstable atherosclerosis that will facilitate the identification and validation of long-sought-after therapeutic targets and drugs for plaque stabilization.