Detection and quantification of protein adduction by electrophilic fatty acids: mitochondrial generation of fatty acid nitroalkene derivatives

TY - JOUR

T1 - Detection and quantification of protein adduction by electrophilic fatty acids: mitochondrial generation of fatty acid nitroalkene derivatives

AU - Schopfer, F. J.

AU - Batthyany, C.

AU - Baker, P. R.S.

AU - Bonacci, G.

AU - Cole, M. P.

AU - Rudolph, V.

AU - Groeger, A. L.

AU - Rudolph, T. K.

AU - Nadtochiy, S.

AU - Brookes, P. S.

AU - Freeman, B. A.

N1 - Funding Information: This work was supported in part by National Institutes of Health Grants HL58115 and HL64937 (to B.A.F.), AHA 0665418U (to F.J.S.) and ADA 7-08-JF-52 (to F.J.S.), ADA 7-06-JF-06 (to P.R.S.B), P30 DK046204-15 and T32DK007052-34 (to M.C.P.), and the Deutsche Herzstiftung (to V.R. and T.K.R.). B.A.F. acknowledges a financial interest in Complexa, LLC. We thank Eric Kelley, Ph.D., and Bruce Branchaud, Ph.D., for helpful guidance.

PY - 2009/5/1

Y1 - 2009/5/1

N2 - Nitroalkene fatty acid derivatives manifest a strong electrophilic nature, are clinically detectable, and induce multiple transcriptionally regulated anti-inflammatory responses. At present, the characterization and quantification of endogenous electrophilic lipids are compromised by their Michael addition with protein and small-molecule nucleophilic targets. Herein, we report a trans-nitroalkylation reaction of nitro-fatty acids with β-mercaptoethanol (BME) and apply this reaction to the unbiased identification and quantification of reaction with nucleophilic targets. Trans-nitroalkylation yields are maximal at pH 7 to 8 and occur with physiological concentrations of target nucleophiles. This reaction is also amenable to sensitive mass spectrometry-based quantification of electrophilic fatty acid-protein adducts upon electrophoretic resolution of proteins. In-gel trans-nitroalkylation reactions also permit the identification of protein targets without the bias and lack of sensitivity of current proteomic approaches. Using this approach, it was observed that fatty acid nitroalkenes are rapidly metabolized in vivo by a nitroalkene reductase activity and mitochondrial β-oxidation, yielding a variety of electrophilic and nonelectrophilic products that could be structurally characterized upon BME-based trans-nitroalkylation reaction. This strategy was applied to the detection and quantification of fatty acid nitration in mitochondria in response to oxidative inflammatory conditions induced by myocardial ischemia-reoxygenation.

AB - Nitroalkene fatty acid derivatives manifest a strong electrophilic nature, are clinically detectable, and induce multiple transcriptionally regulated anti-inflammatory responses. At present, the characterization and quantification of endogenous electrophilic lipids are compromised by their Michael addition with protein and small-molecule nucleophilic targets. Herein, we report a trans-nitroalkylation reaction of nitro-fatty acids with β-mercaptoethanol (BME) and apply this reaction to the unbiased identification and quantification of reaction with nucleophilic targets. Trans-nitroalkylation yields are maximal at pH 7 to 8 and occur with physiological concentrations of target nucleophiles. This reaction is also amenable to sensitive mass spectrometry-based quantification of electrophilic fatty acid-protein adducts upon electrophoretic resolution of proteins. In-gel trans-nitroalkylation reactions also permit the identification of protein targets without the bias and lack of sensitivity of current proteomic approaches. Using this approach, it was observed that fatty acid nitroalkenes are rapidly metabolized in vivo by a nitroalkene reductase activity and mitochondrial β-oxidation, yielding a variety of electrophilic and nonelectrophilic products that could be structurally characterized upon BME-based trans-nitroalkylation reaction. This strategy was applied to the detection and quantification of fatty acid nitration in mitochondria in response to oxidative inflammatory conditions induced by myocardial ischemia-reoxygenation.

KW - Electrophiles

KW - Fatty acids

KW - Free radicals

KW - Michael addition

KW - Nitrated fatty acid

KW - Nitroalkenes

KW - Protein adduction

UR - http://www.scopus.com/inward/record.url?scp=63349104334&partnerID=8YFLogxK

U2 - 10.1016/j.freeradbiomed.2008.12.025

DO - 10.1016/j.freeradbiomed.2008.12.025

M3 - SCORING: Journal article

C2 - 19353781

AN - SCOPUS:63349104334

VL - 46

SP - 1250

EP - 1259

JO - FREE RADICAL BIO MED

JF - FREE RADICAL BIO MED

SN - 0891-5849

IS - 9

ER -