title: Assay of protein and peptide adducts of cholesterol ozonolysis products by hydrophobic and click enrichment methods.
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 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 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.
authors.affiliation: Department of Chemistry, ‡Vanderbilt Institute of Chemical Biology, and ∥Vanderbilt Kennedy Center for Research on Human Development, Vanderbilt University , Nashville, Tennessee 37235, United States.
authors.forename: Katherine
authors.initials: K
authors.lastname: Windsor
authors.forename: Thiago C
authors.initials: TC
authors.lastname: Genaro-Mattos
authors.forename: Sayuri
authors.initials: S
authors.lastname: Miyamoto
authors.forename: Donald F
authors.initials: DF
authors.lastname: Stec
authors.forename: Hye-Young H
authors.initials: HY
authors.lastname: Kim
authors.forename: Keri A
authors.initials: KA
authors.lastname: Tallman
authors.forename: Ned A
authors.initials: NA
authors.lastname: Porter
mesh_terms: D000447:Aldehydes / Q000737:chemistry; D000595:Amino Acid Sequence; D001710:Biotin / Q000737:chemistry; D002784:Cholesterol / Q000031:analogs & derivatives / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D057930:Click Chemistry; D045304:Cytochromes c / Q000737:chemistry; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007536:Isomerism; D008969:Molecular Sequence Data; D010126:Ozone / Q000737:chemistry*; D010455:Peptides / Q000032:analysis / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D012709:Serum Albumin / Q000737:chemistry; D052616:Solid Phase Extraction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019809:Streptavidin / Q000737:chemistry
chemical_list: D000447:Aldehydes; D010455:Peptides; D011506:Proteins; D012709:Serum Albumin; C002415:cholesterol alpha-oxide; D010126:Ozone; D001710:Biotin; D045304:Cytochromes c; D019809:Streptavidin; D002784:Cholesterol