Abstract
The aging process seems to be associated with oxidative stress and hence increased production of lipid peroxidation products, including isolevuglandins (isoLGs). The latter are highly reactive γ-ketoaldehydes which can form covalent adducts with primary amino groups of enzymes and proteins and alter the properties of these biomolecules. Yet little is currently known about amino acid-containing compounds affected by isoLG modification in different age-related pathological processes. To facilitate the detection of these biomolecules, we developed a strategy in which the purified enzyme (or protein) of interest is first treated with authentic isoLG in vitro to evaluate whether it contains reactive lysine residues prone to modification with isoLGs. The data obtained serve as a basis for making the “GO/NO GO” decision as to whether to pursue a further search of this isoLG modification in a biological sample. In this chapter, we describe the conditions for the in vitro isoLG modification assay and how to use mass spectrometry to identify the isoLG-modified peptides and amino acid residues. Our studies were carried out on cytochrome P450 27A1, an important metabolic enzyme, and utilized iso[4]levuglandin E2 as a prototypical isoLG. The isoLG-treated cytochrome P450 was subjected to proteolysis followed by liquid chromatography-tandem mass spectrometry for peptide separation and analysis by Mascot, a proteomics search engine, for the presence of modified peptides. The developed protocol could be applied to characterization of other enzymes/proteins and other types of unconventional posttranslational protein modification.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Salomon RG, Miller DB (1985) Levuglandins: isolation, characterization, and total synthesis of new secoprostanoid products from prostaglandin endoperoxides. Adv Prostaglandin Thromboxane Leukot Res 15:323–326
Salomon RG, Miller DB, Zagorski MG et al (1984) Prostaglandin endoperoxides. 14. Solvent-induced fragmentation of prostaglandin endoperoxides. New aldehyde products from PGH2 and a novel intramolecular 1,2-hydride shift during endoperoxide fragmentation in aqueous solution. J Am Chem Soc 106:6049–6060
Miller DB, Raychaudhuri SR, Avasthi K et al (1990) Prostaglandin endoperoxides. 25. Levuglandin E2: enantiocontrolled total synthesis of a biologically active rearrangement product from the prostaglandin endoperoxide PGH2. J Org Chem 55:3164–3175
Salomon RG, Subbanagounder G, Singh U et al (1997) Oxidation of low-density lipoproteins produces levuglandin-protein adducts. Chem Res Toxicol 10:750–759
DiFranco E, Subbanagounder G, Kim S et al (1995) Formation and stability of pyrrole adducts in the reaction of levuglandin E2 with proteins. Chem Res Toxicol 8:61–67
Salomon RG, Sha W, Brame C et al (1999) Protein adducts of iso[4]levuglandin E2, a product of the isoprostane pathway, in oxidized low density lipoprotein. J Biol Chem 274:20271–20280
Salomon RG (2005) Distinguishing levuglandins produced through the cyclooxygenase and isoprostane pathways. Chem Phys Lipids 134:1–20
Kobierski ME, Kim S, Murthi KK et al (1994) Synthesis of a pyrazole isostere of pyrroles formed by the reaction of the .epsilon.-amino groups of protein lysyl residues with levuglandin E2. J Org Chem 59:6044–6050
Li W, Laird JM, Lu L et al (2009) Isolevuglandins covalently modify phosphatidylethanolamines in vivo: detection and quantitative analysis of hydroxylactam adducts. Free Radic Biol Med 47:1539–1552
Poliakov E, Brennan ML, Macpherson J et al (2003) Isolevuglandins, a novel class of isoprostenoid derivatives, function as integrated sensors of oxidant stress and are generated by myeloperoxidase in vivo. FASEB J 17:2209–2220
Salomon RG (2005) Levuglandins and isolevuglandins: stealthy toxins of oxidative injury. Antioxid Redox Signal 7:185–201
Salomon RG, Kaur K, Batyreva E (2000) Isolevuglandin-protein adducts in oxidized low density lipoprotein and human plasma: a strong connection with cardiovascular disease. Trends Cardiovasc Med 10:53–59
Govindarajan B, Laird J, Salomon RG et al (2008) Isolevuglandin-modified proteins, including elevated levels of inactive calpain-1, accumulate in glaucomatous trabecular meshwork. Biochemistry (Mosc) 47:817–825
Zagol-Ikapitte I, Masterson TS, Amarnath V et al (2005) Prostaglandin H(2)-derived adducts of proteins correlate with Alzheimer’s disease severity. J Neurochem 94:1140–1145
Fukuda K, Davies SS, Nakajima T et al (2005) Oxidative mediated lipid peroxidation recapitulates proarrhythmic effects on cardiac sodium channels. Circ Res 97:1262–1269
Stavrovskaya IG, Baranov SV, Guo X et al (2010) Reactive gamma-ketoaldehydes formed via the isoprostane pathway disrupt mitochondrial respiration and calcium homeostasis. Free Radic Biol Med 49:567–579
Charvet C, Liao WL, Heo GY et al (2011) Isolevuglandins and mitochondrial enzymes in the retina: mass spectrometry detection of post-translational modification of sterol-metabolizing CYP27A1. J Biol Chem 286:20413–20422
Charvet CD, Laird J, Xu Y et al (2013) Posttranslational modification by an isolevuglandin diminishes activity of the mitochondrial cytochrome P450 27A1. J Lipid Res 54:1421–1429
Pikuleva IA (2006) Cytochrome P450s and cholesterol homeostasis. Pharmacol Ther 112:761–773
Heo G-Y, Bederman I, Mast N et al (2011) Conversion of 7-ketocholesterol to oxysterol metabolites by recombinant CYP27A1 and retinal pigment epithelial cells. J Lipid Res 52:1117–1127
Omarova S, Charvet CD, Reem RE et al (2012) Abnormal vascularization in mouse retina with dysregulated retinal cholesterol homeostasis. J Clin Invest 122:3012–3023
Subbanagounder G, Salomon RG, Murthi KK et al (1997) Total synthesis of Iso[4]-levuglandin E2. J Org Chem 62:7658–7666
Pikuleva IA, Bjorkhem I, Waterman MR (1997) Expression, purification, and enzymatic properties of recombinant human cytochrome P450c27 (CYP27). Arch Biochem Biophys 343:123–130
Shevchenko A, Tomas H, Havlis J et al (2007) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1:2856–2860
Lee MS (2012) Mass spectrometry handbook. Wiley, Hoboken, NJ
Acknowledgements
We thank the National Institutes of Health (the EY018383 grant to I.A.P. and the T32 EY007157 fellowship to C.D.C.). I.A.P. is the recipient of a Jules and Doris Stein Professorship from the Research to Prevent Blindness. We are grateful to Robert G. Salomon for his enthusiastic collaboration.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Charvet, C.D., Pikuleva, I.A. (2015). Mass Spectrometry Detection of Isolevuglandin Adduction to Specific Protein Residues. In: Armstrong, D. (eds) Advanced Protocols in Oxidative Stress III. Methods in Molecular Biology, vol 1208. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1441-8_21
Download citation
DOI: https://doi.org/10.1007/978-1-4939-1441-8_21
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-1440-1
Online ISBN: 978-1-4939-1441-8
eBook Packages: Springer Protocols