A TANDEM MS PRECURSOR-ION SCAN APPROACH FOR THE IDENTIFICATION OF VARIABLE COVALENT MODIFICATION OF ALBUMIN CYS34 TO STUDY VASCULAR CARBONYLATION

To date, several experimental evidences confirm that protein carbonylation induced by reactive carbonyl species (RCS) is involved in the pathogenesis of atherosclerosis and subsequently in the development of CVD. Hence, an analytical approach aimed to identify and characterize vascular RCS would be an important tool for the following goals: (i) to better understand the pathogenetic role of RCS, (ii) to monitor and predict the progression of the disease, (iii) to identify novel drug target, and (iv) to demonstrate the efficacy of carbonyl sequestering agents. We recently found that human serum albumin (HSA) is a detoxyfing vascular protein of reactive carbonyl species (RCS) through a covalent adduction mechanism, and that Cys34 is the most reactive site, giving the corresponding Michael adduct [1]. Since Cys34 is one of the main vascular targets of RCS, the identification and characterization of the covalent Cys34 modifications would be a powerful tool to study vascular carbonylation and to reach the goal above mentioned. To do this, we set-up a mass spectrometric approach that permits to identify unknown covalent modifications of Cys34 based on a triple quadrupole mass spectrometer in pre-cursor ion scan mode. In particular, by considering the trypsin/chymotrypsin digested peptide containing Cys34 (LQQCPF), the y and b fragment which precedes and follows the modification site are selected as precursor ions. The data analysis by using an algorithm developed by us, permits a rapid and specific search of the precursor-ions series attributed to native and modified target peptide. In a following step, the identified pre-cursor ions are selected for product ion scan analysis in order to identify and characterize the type of adduction. The method has been validated by using 4-hydroxy-trans-nonenal (HNE) as a RCS model. Human serum was incubated in the presence of HNE (10 nmoles ml-1), then albumin isolated by affinity chromatography and digested by using trypsin/chymotrypsin; the m/z 370.1 (b3) and m/z 263.1 (y2) fragment ions were used as precursor-ions. The approach identified two peaks relative to the LQQCPF native peptide at m/z 792.4 and the corresponding Cys34-HNE adduct at m/z 893.3, as confirmed by MS/MS. In summary the tandem MS approach here reported is a powerful tool for the rapid identification of unknown covalent protein modifications. References 1. Aldini G. et al.; J. Mass Spectrom., 41, 1149-61 (2006)