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Electrospray MS/MS reveals extensive and nonspecific oxidation of cholesterol esters in human peripheral vascular lesions.

ABSTRACT: Although LDL is rendered proatherogenic by various experimental treatments (e.g., acetylation), the exact structural changes that drive LDL transformation in vivo remain enigmatic. Among the many hypothesized targets of oxidative modification are cholesterol esters (CE). This family of neutral lipids, which carries a highly unsaturated pool of fatty acyl groups, is the main component of both LDL particles and atherosclerotic plaques. Tandem mass spectrometry (MS/MS) was employed to reveal abundant and diverse oxidized CEs (oxCE), including novel oxidation products, within human peripheral vascular lesions. These oxCE species composed up to 40% of the total CE pool, with cholesteryl linoleate being oxidized to the greatest extent. Imaging mass spectrometry studies showed that oxCE was entirely confined within the plaque, along with unmodified CE and triacylglyceride (TAG). Interestingly, we found no evidence for TAG oxidation, although polyunsaturated species were abundant. Enzymatic oxidation of cholesteryl linoleate by 15-lipoxygenase (15-LO), an enzyme often invoked in CE oxidation, initially results in a regio- and stereospecific product. Analysis of intact cholesteryl hydroxyoctadecadienoate isomers in human atheromata revealed no regio- or stereospecificity, indicating 15-LO was either not a major source of oxCE or nonenzymatic processes had eroded any product specificity.

SUBMITTER: Hutchins PM

PROVIDER: S-EPMC3196238 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Electrospray MS/MS reveals extensive and nonspecific oxidation of cholesterol esters in human peripheral vascular lesions.

Hutchins Patrick M PM Moore Ernest E EE Murphy Robert C RC

Journal of lipid research 20110831 11

Although LDL is rendered proatherogenic by various experimental treatments (e.g., acetylation), the exact structural changes that drive LDL transformation in vivo remain enigmatic. Among the many hypothesized targets of oxidative modification are cholesterol esters (CE). This family of neutral lipids, which carries a highly unsaturated pool of fatty acyl groups, is the main component of both LDL particles and atherosclerotic plaques. Tandem mass spectrometry (MS/MS) was employed to reveal abunda ...[more]

PMID: 21885431

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Project description:The selective reversible S-glutathiolation of specific SERCA (sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase) cysteine residues represents a novel physiologic pathway of NO (nitric oxide)-dependent arterial smooth muscle relaxation [Adachi, Weisbrod, Pimentel, Ying, Sharov, Schöneich and Cohen (2004) Nat. Med. 10, 1200-1207]. This mechanism may be impaired through the irreversible oxidation of functionally important cysteine residues as a consequence of oxidative stress and aging. To establish whether in vivo aging and in vitro oxidation by peroxynitrite result in the loss of such functionally important cysteine residues of SERCA, we have developed and optimized a quantitative method to monitor the oxidation state of the individual SERCA cysteine residues using a maleimide-based fluorescence dye, TG1 (ThioGlo 1), as a label for cysteine residues that have not been altered by oxidation and are not involved in disulphide bridges. A high efficiency for TG1 labelling of such residues and the chemical structure of cysteine-TG1 adducts were validated by MS analysis of model peptides, model proteins and rat skeletal muscle SERCA1. Tryptic peptides containing 18 out of a total of 24 cysteine residues were identified by HPLC-ESI (electrospray ionization)-MS/MS (tandem MS). Two cysteine residues, at positions 344 and 349, were detected in the form of an internal disulphide bridge, and another 16 were found to be labelled with TG1. Using HPLC-ESI-MS, we quantitatively mapped peroxynitrite oxidation of eight cysteine residues (positions 364, 417, 420, 498, 525, 674, 675 and 938), some of which are involved in the control of SERCA activity. Biological aging resulted in the partial modification of cysteine residues 377, 498, 525, 561, 614, 636, 674, 675, 774 and 938. Neither peroxynitrite exposure nor biological aging affected the apparent SERCA1 ATP affinity. Our data show an age-dependent loss of cysteine residues (approx. 2.8 mol of cysteine/mol of SERCA1), which may be partially responsible for the age-dependent decrease in the specific Ca2+-ATPase activity (by 40%).

Dataset Information

Electrospray MS/MS reveals extensive and nonspecific oxidation of cholesterol esters in human peripheral vascular lesions.

Publications

Electrospray MS/MS reveals extensive and nonspecific oxidation of cholesterol esters in human peripheral vascular lesions.

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