Assay of Protein and PeptideAdducts of CholesterolOzonolysis Products by Hydrophobic and Click Enrichment Methods

摘要

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 amino 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 hydrophobicenrichment method with proteomic analysis utilizing characteristicfragmentation patterns facilitates the identification of secosterol-modifiedpeptides and proteins in an adducted protein. More broadly, theseimproved enrichment methods may give insight into the role of oxysterolsand ozone exposure in the pathogenesis of a variety of diseases, includingatherosclerosis, Alzheimer’s disease, Parkinson’s disease,and asthma.