Assay of protein and peptide adducts of cholesterol ozonolysis products by hydrophobic and click enrichment methods

Katherine Windsor, Thiago Cardoso Genaro De Mattos, Sayuri Miyamoto, Donald F. Stec, Hye Young H. Kim, Keri A. Tallman, Ned A. Porter

Research output: Contribution to journalArticle

7 Citations (Scopus)

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. (Chemical Equation Presented)

Original languageEnglish (US)
Pages (from-to)1757-1768
Number of pages12
JournalChemical Research in Toxicology
Volume27
Issue number10
DOIs
StatePublished - Oct 20 2014

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Assays
Cholesterol
Peptides
Proteins
Amino Acids, Peptides, and Proteins
Ozone
Bioactivity
Dehydration
Aldehydes
Solid Phase Extraction
Oxysterols
Proteomics
Parkinson Disease
Atherosclerosis
Alzheimer Disease
Derivatives
Asthma

ASJC Scopus subject areas

  • Toxicology

Cite this

Assay of protein and peptide adducts of cholesterol ozonolysis products by hydrophobic and click enrichment methods. / Windsor, Katherine; Cardoso Genaro De Mattos, Thiago; Miyamoto, Sayuri; Stec, Donald F.; Kim, Hye Young H.; Tallman, Keri A.; Porter, Ned A.

In: Chemical Research in Toxicology, Vol. 27, No. 10, 20.10.2014, p. 1757-1768.

Research output: Contribution to journalArticle

Windsor, Katherine ; Cardoso Genaro De Mattos, Thiago ; Miyamoto, Sayuri ; Stec, Donald F. ; Kim, Hye Young H. ; Tallman, Keri A. ; Porter, Ned A. / Assay of protein and peptide adducts of cholesterol ozonolysis products by hydrophobic and click enrichment methods. In: Chemical Research in Toxicology. 2014 ; Vol. 27, No. 10. pp. 1757-1768.
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