Top-Down Proteomics of HDL Particles in Atherosclerosis
In collaboration with the proposed NRTDP, we will expand on the Wilkins laboratory’s research and involvement with a regional and national group of scientists studying heart disease in America (the Multi-Ethnic Study of Atherosclerosis (MESA) and the Coronary Artery Risk Development in Young Adults Study (CARDIA) studies, both with Field Centers in Chicago, IL) to create a driving biomedical project whose goal is to characterize the intact proteome of the lipoprotein particles. Such analysis is critical, as these proteins undergo extensive processing, and there are preliminary data that show a particularly strong correlation of apolipoprotein C-III proteoforms to HDL/LDL levels . Apolipoprotein C-III and ApoA-I have been associated with cardiovascular heart disease (CHD) and their various proteoforms might demonstrate correlations in discrimination of individuals in a population study. After characterization and quantification, CHD-related proteoforms that correlate with “extreme phenotypes” (i.e., individuals with very high or low risk for heart events) will be sought as described in DBP 3. Even now, just 10 μL of serum or an equivalent amount of blood have been used to extract lipoproteins for LC-MS to identify and characterize lipoprotein proteoforms by reverse phase liquid chromatography coupled to FT Orbitrap MS in a targeted fashion.
Using the improved LC-MS techniques developed by the NRTDP in TR&D 1 and the new instruments designed for top-down mass spectrometry (TR&D 2), we will characterize specific variations and any unexpected protein processing steps that take place within the human population measured. The numbers of longitudinal samples archived through these national cohorts can become quite large. Therefore, in this DBP we will first employ discovery experiments (Task 1) where proteoforms of interest are characterized and catalogued. Subsequent to this will be a larger-scale profiling experiment (Task 2). Consequently, top-down proteomics will provide valuable insights into the range of proteoforms in human heart wellness, as well as any differences seen in atherosclerosis.