Enzymatic and non-enzymatic lipid metabolism can give rise to reactive species that may covalently modify cellular or plasma proteins through a process known as lipoxidation. Under basal conditions, protein lipoxidation can contribute to normal cell homeostasis and participate in signaling or adaptive mechanisms, as exemplified by lipoxidation of Ras proteins or of the cytoskeletal protein vimentin, both of which behave as sensors of electrophilic species. Nevertheless, increased lipoxidation under pathological conditions may lead to deleterious effects on protein structure or aggregation. This can result in impaired degradation and accumulation of abnormally folded proteins contributing to pathophysiology, as may occur in neurodegenerative diseases. Identification of the protein targets of lipoxidation and its functional consequences under pathophysiological situations can unveil the modification patterns associated with the various outcomes, as well as preventive strategies or potential therapeutic targets. Given the wide structural variability of lipid moieties involved in lipoxidation, highly sensitive and specific methods for its detection are required. Derivatization of reactive carbonyl species is instrumental in the detection of adducts retaining carbonyl groups. In addition, use of tagged derivatives of electrophilic lipids enables enrichment of lipoxidized proteins or peptides. Ultimate confirmation of lipoxidation requires high resolution mass spectrometry approaches to unequivocally identify the adduct and the targeted residue. Moreover, rigorous validation of the targets identified and assessment of the functional consequences of these modifications are essential. Here we present an update on methods to approach the complex field of lipoxidation along with validation strategies and functional assays illustrated with well-studied lipoxidation targets.
Bibliographical note© 2015 Published by Elsevier B.V.
Funding: MINECO SAF2012-36519 and ISCIII RETIC RD12/0013/0008 (Spain) to DPS; PEst-C/QUI/UI0062/2013, FCOMP-01-0124-FEDER-037296 to QOPNA research unit and RNEM-REDE/1504/REM/2005 to Portuguese National Mass Spectrometry Network by Fundação para a Ciência e a Tecnologia (FCT, Portugal), European Union, QREN, FEDER, and COMPETE to MRMD and PD. CMS acknowledges funding from the Engineering and Physical Sciences Research
Council, UK, EP/I017887/1 Cross-Disciplinary Research Landscape Award (the Proxomics Project). Collaboration among the authors’ laboratories has been supported by EU COST Action CM1001 on “Chemistry of non-enzymatic protein modification - modulation of protein structure and function”
- cyclopentenone prostaglandins
- electrophilic lipids
- mass spectrometry
- reactive carbonyl species
- target validation
- vimentin cysteine lipoxidation