Study of protein modification by 4-hydroxy-2-nonenal and other short chain aldehydes analyzed by electrospray ionization tandem mass spectrometry

Abstract

A convenient way to study lipid oxidation products-modified proteins by means of suitable model systems has been investigated. As a model peptide, the oxidized B chain of insulin has been chemically modified by either 4-hydroxy-2-nonenal (HNE) or hexanal and the extent, sites, and structure of modifications were assessed by electrospray mass spectrometry. A reduction step, using either NaCNBH 3 or NaBH 4 , was also studied to stabilize the alkylated compounds. From the data gathered, it appeared that NaCNBH 3 , when added at the beginning of incubation, dramatically influenced the HNE-induced modifications in terms of the addition mechanism (Schiff base formation instead of Michael addition) but also of the amino acid residues modified (N-terminal amino acid instead of histidine residues). However, by reducing the HNE-adducted species at the end of the reaction with NaBH 4 , the fragment ions obtained in the product ion scan experiments become more stable and thus, easier to interpret in terms of origin and mechanism involved. With regard to hexanal induced modifications, we have observed that hexanal addition under reductive conditions led to an extensive modification of the peptide backbone. Moreover, as confirmed by "in-source" collision followed by collision induced dissociation (CID) experiments on selected precursor ions (pseudo-MS 3 experiments), N,N-di-alkylations were first observed on the N-terminal residue and further on Lys 29 residue. On the other hand, compared to the native peptide, no significant changes in MS/MS fragmentation patterns (b and y ions series) were observed whatever the basic site modified by the aldehyde-addition. (J Am Soc Mass Spectrom 2003, 14, 215-226)

Membrane-mediated Amyloidogenesis and the Promotion of Oxidative Lipid Damage by Amyloid β Proteins

“…3. However, numerous attempts to detect Schiff base formation in various proteins have concluded that the extent to which HNE modifies Lys side chains is insignificant compared with the extent to which His side chains are modified (25,47,(61)(62)(63)(64)(65)(66). Moreover, immunohistochemical studies have shown that anti-HNE-His antibodies bind to amyloid plaques in AD brain tissue (26), but anti-HNE-Lys antibodies do not (36).…”

Modification of Cytochrome c by 4-hydroxy- 2-nonenal: Evidence for histidine, lysine, and arginine-aldehyde adducts

“…4HNE-modified lysines have also been characterized in both Schiff base and 2-pentylpyrrole forms [22,23,33]. A Michael addition to lysine or histidine residue results in a mass shift of 156.…”

Post-translational Modifications and Mass Spectrometry Detection

“…Most of the formed adducts are chemically reversible and only those which prove to be stable under the experimental conditions adopted will be detected. A common analytical approach includes a stabilization step by reduction with borohydride (NaBH 4 ) prior to protein digestion (240). MS studies with non-reduced peptide adducts have demonstrated that the major product ions upon tandem MS fragmentation result from the neutral loss of the aldehyde residues, difficulting the assignment of the modification site (241,242).…”