Significance
HDL, a complex matrix composed of lipids and proteins, is implicated in cardioprotection. Its cholesterol content correlates inversely with cardiovascular disease and it is the current metric to assess cardiovascular risk. However, the cholesterol content does not capture HDL's complexity and heterogeneity. Devising metrics that better capture HDL's cardioprotective effects, we developed an optimized method for quantification of HDL proteome, using PRM in concert with a single labeled protein as internal standard. The availability of a method that increases sample throughput without compromising the reproducibility, sensitivity, and accuracy could therefore point to better risk assessment for CVD or other diseases.