A tandem MS precursorion scan approach to identify variable covalent modification of albumin Cys34: a new tool for studying vascular carbonylation

We developed a liquid chromatography electrospray ionisation multistage mass spectrometry LCESIMSMS approach based on precursorion scanning and evaluated it to characterize the covalent modifications of Cys34 human serum albumin HSA caused by oxidative stress and reactive carbonyl species RCS adduction. HSA was isolated and digested enzymatically to generate a suitablelength peptide LQQCPF containing the modified tag residue. The resulting LQQCPF peptides were identified by LCESIMSMS in precursorion scan mode and further characterized in production scan mode. The product ions for precursorion scanning were selected by studying the MSMS fragmentation of a series of LQQCPF derivatives containing Cys34 modified with different α,βunsaturated aldehydes and di and ketoaldehydes. We used a Boolean logic to enhance the specificity of the method: this reconstitutes a virtual current trace vCT showing the peaks in the three precursorion scans, marked by the same parent ion. The method was first evaluated to identify and characterize the Cys34 covalent adducts of HSA incubated with 4hydroxyhexenal, 4hydroxytrans2nonenal HNE and acrolein ACR. Then we studied the Cys34 modification of human plasma incubated with mildly oxidized lowdensity lipoproteins LDL, and the method easily identified the LQQCPF adducts with HNE and ACR. In other experiments, plasma was oxidized by 2,2azobis2amidinopropane HCl AAPH or by Fe2H2O2. In both conditions, the sulfinic derivative of LQQCPF was identified and characterized, indicating that the method is suitable not only for studying RCSmodified albumin, but also to check the oxidative state of Cys34 as a marker of oxidative damage. Copyright © 2008 John Wiley & Sons, Ltd.