A mass spectrometry approach for the identification and localization of lysozyme modifications by acrolein

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Lipids containing polyunsaturated fatty acids are primary targets of oxidation, leading to the formation of a variety of reactive products (1). The new oxidised products are generated with different number of oxygen molecules bound to the acyl chain, increasing the lipid molecular weight. However, the alkoxyl radicals can generate short-chain oxidation products including short-chain aldehydes, which can covalently modify proteins in a process called lipoxidation (2). Acrolein, with only three carbons, is the shortest alkenal identified as a short-chain oxidation product. It is also by far the strongest electrophile and therefore the most reactive, especially with thiol groups on proteins (3). These oxidative post-translational modifications influence cell behaviour and can be involved in inflammatory diseases (4). The exact nature of many of these adducts, and their relationship with cellular effects are still unclear. There is also a need to develop sensitive mass spectrometry (MS) methods that are well-adapted for the identification of these adducts in complex biological systems. The aim of this work was to develop a mass spectrometry approach for the analysis of protein-aldehyde adducts. Lysozyme from chicken egg white was used to investigate the formation of short-chain aldehyde-containing lipoxidation products. The protein was first reduced with dithiothreitol to break disulphide bounds between cysteine residues, generating free thiols available for modification. Acrolein was then added to modify the reduced protein. The protein-aldehyde adducts formed were stabilized by reduction with sodium borohydride and identified using mass spectrometry to analyse the intact protein and tryptic digests. Analysis of intact acrolein-modified lysozyme showed that multiple sites (up to 8) could be modified. All modifications were found to be Michal adducts, increasing the protein mass by 58 Da per adduct. Analysis of tryptic digests allowed the localization of the adducts to specific amino acid residues. Six cysteine and two lysine residues were shown to be modified, which corroborates observation of up to 8 acrolein adducts per protein in the intact protein analysis. By the same proteomic approach, it was possible to identify amino acid-specific fragmentations that may be helpful in the identification of specific acrolein adducts. In summary, this study can be seen as a model testing mass spectrometry for the analysis of protein-aldehyde adducts. The combined use of direct infusion and LC-MS/MS methods helped to identify the type, the number and the location of acrolein modifications in lysozyme. This shows that MS methods provide a powerful tool for the evaluation of these modifications and further aldehydes are now being investigated. Potentially, this MS approach can be applied to discover biomarkers of adductions in cells and tissues under pathophysiological environments.