Discipline: Biological Sciences
Subcategory: Biochemistry (not Cell and Molecular Biology and Genetics)
Room: Exhibit Hall A
Leah Rowe - University of Arkansas at Pine Bluff
Co-Author(s): Linda S. May-Zhang, Vanderbilt University, Nashville,TN; John Melchior, University of Cincinnati, Cincinnati, Ohio; Jamie Morris, University of Cincinnati, Cincinnati, Ohio; W. Sean Davidson, University of Cincinnati, Cincinnati; Sean S. Davies, Vanderbilt University, Nashville, Tn
Cardiovascular disease (CVD) is inversely associated with high density lipoprotein cholesterol (HDL-C), but pharmacological interventions aimed to increase HDL-C have failed to reduce disease risk. Recent evidence suggests that CVD risk more closely relates to HDL function than HDL-C. We have reported that isolevuglandins (IsoLGs), highly reactive lipid dicarbonyls generated by lipid peroxidation, deleteriously alter HDL structure and function, mainly via their modification of apolipoprotein A-1 (apoA-1) lysines. The specific lysines of apoA-1 modified by IsoLG in human HDL in a diseased setting are unknown. This study aims to develop a high-resolution mass spectrometric based method to quantify specific tryptic peptides derived only from IsoLG-modified apoA-I, as well as the unmodified apoA-I peptides. Using reconstituted apoA-I particles, we identified 8 lysine targets of apoA-I by IsoLG. By stable isotope dilution LC/MS/MS with parallel reaction monitoring, we found a dose-dependent increase in IsoLG-lysine adducts in Lys226 with IsoLG modification of human HDL. Lys94, Lys118, and Lys182 adducts were identified at higher IsoLG modification (3 molar eq. to apoA-I). We identified 8 unmodified tryptic apoA-I peptides containing lysines that ?drop-out? from our analyses, suggesting that those peptides are adducted or crosslinked by IsoLG. As a validation of our approach, we were able to detect IsoLG-ApoAI adducts in Lys94, 118, 183, and 226 in oxidized human plasma compared and working on validating clinical samples. Ultimately, our assay will translate to measuring IsoLG adducted apoA-I from plasmas of patients with atherosclerosis, which will give deeper insight into their HDL dysfunction.
Funder Acknowledgement(s): NIH P01 HL-116263 (Linton/Davies); NIH F32 HL138745 (May-Zhang); R25 HL096223-10 (Joyce)
Faculty Advisor: Anissa Buckner, Bucknera@uapb.edu
Role: In this project , I was responsible for the preparation of samples for the mass spec. This included treating the samples with trypsin and verifying that it worked.I also quantified all of the mass spec data via qual browser. Additional assays completed include SDS Page gels and protein assays.