Simultaneous Detection
of Low and High Molecular Weight
Carbonylated Compounds Derived from Lipid Peroxidation by Electrospray
Ionization-Tandem Mass Spectrometry
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Abstract
Reactive oxygen species (ROS) and other oxidative agents
such as
free radicals can oxidize polyunsaturated fatty acids (PUFA) as well
as PUFA in lipids. The oxidation products can undergo consecutive
reactions including oxidative cleavages to yield a chemically diverse
group of products, such as lipid peroxidation products (LPP). Among
them are aldehydes and ketones (“reactive carbonyls”)
that are strong electrophiles and thus can readily react with nucleophilic
side chains of proteins, which can alter the protein structure, function,
cellular distribution, and antigenicity. Here, we report a novel technique
to specifically derivatize both low molecular and high molecular weight
carbonylated LPP with 7-(diethylamino)coumarin-3-carbohydrazide (CHH)
and analyze all compounds by electrospray ionization-mass spectrometry
(ESI-MS) in positive ion mode. CHH-derivatized compounds were identified
by specific neutral losses or fragment ions. The fragment ion spectra
displayed additional signals that allowed unambiguous identification
of the lipid, fatty acids, cleavage sites, and oxidative modifications.
Oxidation of docosahexaenoic (DHA, 22:6), arachidonic (AA, 20:4),
linoleic (LA, 18:2), and oleic acids (OA, 18:1) yielded 69 aliphatic
carbonyls, whose structures were all deduced from the tandem mass
spectra. When four phosphatidylcholine (PC) vesicles containing the
aforementioned unsaturated fatty acids were oxidized, we were able
to deduce the structures of 122 carbonylated compounds from the tandem
mass spectra of a single shotgun analysis acquired within 15 min.
The high sensitivity (LOD ∼ 1 nmol/L for 4-hydroxy-2-nonenal,
HNE) and a linear range of more than 3 orders of magnitude (10 nmol/L
to 10 μmol/L for HNE) will allow further studies on complex
biological samples including plasma