Assay of Protein and Peptide Adducts of Cholesterol Ozonolysis Products by Hydrophobic and Click Enrichment Methods

Author(s)

Windsor, Katherine ^[1] ; Genaro-Mattos, Thiago C. ^{[1, 2]} ; Miyamoto, Sayuri ^[2] ; Stec, Donald F. ^[1] ; Kim, Hye-Young H. ^[1] ; Tallman, Keri A. ^[1] ; Porter, Ned A. ^{[1, 3, 4]}

Affiliation

^[1] Vanderbilt Univ, Dept Chem, Nashville, TN 37235 - USA

^[2] Univ Sao Paulo, Inst Quim, Dept Bioquim, BR-05508070 Sao Paulo - Brazil

^[3] Vanderbilt Univ, Vanderbilt Inst Chem Biol, Nashville, TN 37235 - USA

^[4] Vanderbilt Univ, Vanderbilt Kennedy Ctr Res Human Dev, Nashville, TN 37235 - USA

• Document type: Journal article
• Source: Chemical Research in Toxicology; v. 27, n. 10, p. 1757-1768, OCT 2014.
• Web of Science Citations: 7

Abstract

Cholesterol undergoes ozonolysis to afford a variety of oxysterol products, including cholesterol-5,6-epoxide (CholEp) and the isomeric aldehydes secosterol A (seco A) and secosterol B (seco B). These oxysterols display numerous important biological activities, including protein adduction; however, much remains to be learned about the identity of the reactive species and the range of proteins modified by these oxysterols. Here, we synthesized alkynyl derivatives of cholesterol derived oxysterols and employed a straightforward detection method to establish secosterols A and B as the most protein reactive of the oxysterols tested. Model adduction studies with an ammo acid, peptides, and proteins provide evidence for the potential role of secosterol dehydration products in protein adduction. Hydrophobic separation methods Folch extraction and solid phase extraction (SPE) were successfully applied to enrich oxysterol-adducted peptide species, and LC-MS/MS analysis of a model peptide seco adduct revealed a unique fragmentation pattern (neutral loss of 390 Da) for that species. Coupling a hydrophobic enrichment method with proteomic analysis utilizing characteristic fragmentation patterns facilitates the identification of secosterol-modified peptides and proteins in an adducted protein. More broadly, these improved enrichment methods may give insight into the role of oxysterols and ozone exposure in the pathogenesis of a variety of diseases, including atherosclerosis, Alzheimer's disease, Parkinson's disease, and asthma. (AU)

• FAPESP's process: 12/16145-5 - The use of click chemistry as a tool for studying protein modifications by cholesterol aldehydes
• Grantee: Thiago Cardoso Genaro de Mattos
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate