Identification of free radicals and other oxidation products of biomolecules by mass spectrometry

• Autor(a) principal: Pereira, Ana Luísa dos Reis
• Data de Publicação: 2006
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/3176
• topic: Bioquímica; Biomoléculas; Ácidos gordos; Processos aeróbicos; Radicais livres (Química)

The radical peroxidation of lipids and phospholipids is an increasing topic of research due to the close association between lipid peroxidation products and age-related diseases. In this work, mass spectrometry was used for the identification and characterisation of lipid and phospholipid free radicals using the methodology developed for the detection of free radicals, namely the use of spin traps. For this, radical oxidation was performed on ω-6 unsaturated fatty acid and extended to progressive more complex structures, particularly diacylglycerophosphatidylcholines (GPC) containing ω-9 (oleic) and ω-6 (linoleic and arachidonic) fatty acids. Several intact and breakdown free radicals products of lipid and GPC were identified as spin adducts for the first time, and characterisation by tandem mass spectrometry (MS/MS) in association with reverse-phase chromatography (HPLC-MS/MS) allowed the assignment of such species to be carbon and oxygen centred radicals. The lipid breakdown products suggested that β-scission cleavage is selective towards the formation of the smaller alkyl radicals. Also, the intact free radicals identified for the 3 GPC suggested the occurrence of a “retarder” effect to the lipid peroxidation caused by the addition of the spin trap. The identification and characterisation by MS was extended to non-radical GPC products and the complexity of structures required previous separation method by reverse-phase chromatography prior to mass spectrometry detection (LC-MS) and tandem mass spectrometry (LC-MS/MS). More than 50 different structures were identified for only three phospholipids. Based on the LC-MS/MS data analysis, isomeric structures of long-chain products were differentiated and, short-chain products containing different terminal groups were differentiated by LC-MS data analysis according to the retention times, which was independent of the oxidised GPC. In this way, isobaric structures of short-chain products may be distinguished if present in complex mixtures. Adducts formed by covalent interaction between GPC alkenal products and peptides established in in vitro conditions were characterised by MS/MS. Upon fragmentation, the peptide-GPC adducts provided information about the peptide and the lipid moiety, which is characteristic of peptide-GPC adducts due to charge retention in the polar head. Altogether, MS is a suitable technique for the identification of both radical and non-radical products of fatty acids and phosphatidylcholines. Furthermore, the GPC peroxidation products identified in this study suggested that, during nonenzymatic phospholipid radical oxidation, bis-allylic hydrogen atoms closer to the polar head are preferably abstracted.