Polyunsaturated fatty acids present in membrane lipids are susceptible to free radical-initiated peroxidation.
4-Hydroxy-2-nonenal (HNE), a major product of lipid peroxidation (LPO), is regarded as a highly toxic aldehyde formed during peroxidation of polyunsaturated (n−6) fatty acids such as linoleic acid and arachidonic acid.[1,2]
This aldehyde acts as a toxic second messenger of oxidative stress by disseminating and augmenting initial free-radical events. HNE has been implicated in numerous pathologies, such as atherosclerosis, cancer, neurodegenerative disorders (Alzheimer’s disease, Parkinson’s disease and Amyotrophic lateral sclerosis), ethanol-associated liver injury, and injury from ischemia/reperfusion.[3–5]
HNE, a bifunctional aldehyde, is susceptible to nucleophilic addition at both the double bond (C3) and the carbonyl moiety (C1). The presence of electron-withdrawing carbonyl group at C1 position in HNE makes the double bond at the C3 position highly electrophilic and, hence, can covalently bind to proteins via Michael addition (MA) to the sulfhydryl group of cysteine (Cys, C), imidazole group of histidine (His, H), and ε-amino group of lysine (Lys, K) residues resulting in mass increase of 156 Da ().
The reactivity of these amino acids to HNE occurs in the following order: Cys >> His > Lys >> Arg.
Although the Michael adducts of HNE with Cys and His are stable, Michael adducts to Lys ε-amino groups may form reversibly.
The C1 carbonyl moiety of HNE can also yield Schiff-base adducts with the ε-NH2
group of Lys and a concomitant loss of water produces a gain of 138 Da in molecular mass.
Less abundant Schiff base formation with Lys groups results in some late stage stable adducts such as the 2-pentylpyrroles. The bifunctionality of HNE also allows it to induce protein cross-links such as the 2-hydroxy-2-pentyl-1,2-dihydropyrrol-3-one iminium that are fluorescent four-electron oxidation products.
Figure 1 Primary reaction chemistry observed for 4-hydroxy-2-nonenal (HNE) modification of proteins. The α,β-unsaturated aldehyde is susceptible to Michael addition by a variety of nucleophiles including the histidine (His) imidazole group, lysine (more ...)
Quantification or comparison of relative abundance of proteins expressed in cells or tissues during normal or diseased conditions is essential to identify the putative target proteins correlated with the diseased states. However, not all proteins undergo change in their relative expression profile; instead, they may undergo change in the levels of their posttranslational modification. A wide array of functions is controlled by HNE-mediated protein carbonylation. Several studies have shown that change in protein carbonylation in various diseases occurs without any concomitant variation in the protein expression levels;[11–15]
however, most of these studies have used gel electrophoresis for validation. In these studies, changes in protein carbonylation are determined by comparing the spot intensities upon labeling with antibodies specific for HNE or prior labeling of protein carbonyl groups with digoxigenin-hydrazide
and detecting by anti-digoxigenin or anti-2,4-dinitrophenol antibodies in immunoblots obtained from different samples. An additional method involves biotinylation of oxidized proteins and detecting them in two-dimensional (2D-) gels using avidin-fluorescein isothiocyanate staining.
The protein identity in the spot or band is then determined by excising it from the gel, performing enzymatic digestion, and analyzing by mass spectrometry.
The 2D-gel-based method is labor-intensive and time-consuming which confines high-throughput proteome analysis. The low specificity of anti-HNE antibodies and the lack of site-specific HNE antibodies that recognize a particular carbonylation site within a protein sequence make this type of quantification challenging. These limitations have accentuated the need for a technique that can quantify changes in the relative carbonylation states in proteins along with identification of the modification sites.
Mass spectrometry has an unprecedented advantage in protein identification and determination of posttranslational modifications. Several strategies have also been reported to quantitate differential protein expression in tissues.
Extending beyond our previous studies that involve the development of methods for the determination of HNE modification,[18–20]
we describe here an approach that incorporates stable-isotope dimethyl labeling of N-terminus and ε-amino side chains of lysine residues of the peptides for differential quantification of HNE-modified protein. The applicability of stable-isotope dimethyl labeling for quantitative protein profiling has been reported previously.[21,22]
The corresponding stable-isotope labeling allows samples to be analyzed and compared in a single analytical experiment, and does not require the use of labeled peptide standards such as the so-called absolute quantification approach.
The peptide amines are differentially labeled via reductive alkylation with isotopically coded formaldehydes (d0
C-formaldehyde or d2
C-formaldehyde). Following reductive methylation of primary amino groups, each isotopic peptide pair differs in mass by n-times 6 Da, where n is the number of primary amino groups in the peptide. This provides sufficient spacing by m/z
without overlap of the isotope envelopes, facilitating the measurement of relative abundances of peptides by LC–MS. For carbonylated peptides that are normally present in extremely minute amounts, enrichment techniques to minimize interference, such as ion suppression effects caused by large amounts of non-carbonylated peptides,
are normally necessary. Hence, we have incorporated a chemoprecipitation method to selectively isolate HNE-modified peptides and thereby minimize such ion suppression effects from unmodified peptides. The enriched fraction is then subjected to LC–MS and the quantification between the relative abundance of isotopically labeled populations of peptide carbonyls can be computed from comparative area measurements using extracted ion chromatograms (XICs) of light- and heavy-isotopic dimethyl-labeled peptides, respectively. The implementation of a quantification method for protein carbonyls in a biological system is expected to further our understanding of disease mechanisms associated with oxidative stress and, thus, assist the discovery of novel therapeutics for these conditions.[5,25]