Human serum albumin (HSA) was subjected to oxidative stress and the locations of the resulting protein carbonyls were determined using mass spectrometry in conjunction with a hydrazide labeling scheme. To model oxidative stress, HSA samples were subjected to metal-catalyzed oxidation (MCO) conditions or treated with hypochlorous acid (HOCl). Oxidation led to the conversion of lysine residues to 2-aminoadipic semi-aldehyde residues, which were subsequently labeled with biotin hydrazide. Analysis of the tryptic peptides from the samples indicates that the oxidations are highly selective. Under MCO conditions, only two of the 59 lysine residues appeared to be modified (Lys-97 and Lys-186). With HOCl, five different lysine modification sites were identified . These results strongly suggest that the preferred site of modification is dependent on the nature of the oxidant and that the process relies on specific structural motifs in the protein to direct the oxidation. The high selectivity seen here provides insights into the factors that in vivo drive the selective carbonylation of specific proteins in systems under oxidative stress. ( t is well established that oxidative stress leads to a variety of protein modifications including carbonylation [1][2][3][4]. In particular, protein carbonyls have been identified as key biomarkers for disease [5][6][7] and aging [8 -11]. Oxidation of the side-chain functional groups of lysine (Scheme 1), arginine, and proline to aldehyde groups (as well as threonine to a ketone) are common means of generating protein carbonyls [12,13]. Studies by a number of workers, including Sohal [14 -17], have shown that protein carbonylation is selective and that only a subset of proteins is modified in systems under oxidative stress [18 -26]. The underlying reasons behind this selectivity are not well understood. Although protein carbonylation has been characterized in numerous systems under a variety of conditions [27][28][29], these studies only have targeted total carbonyl levels and the distribution between bound and free carbonyl species [30]. Only recently have data emerged on the specific sites of carbonylation in proteins subjected to oxidative stress [31][32][33]. The best data come from recent work by Mirzaei and Regnier [31,32], who have identified carbonylation sites in proteins isolated from yeast and rats subjected to in vivo oxidative stress and in model protein oxidation systems. The sites of protein carbonylation represents an important issue because the factors that govern the selective carbonylation of proteins in vivo are likely to be manifested in the carbonylation site selectivity of a given protein. It is our hypothesis that the carbonylation of a protein is site selective and that structural features in the protein will determine the preferred sites of carbonylation.Although headway has been made in identifying the sites of other oxidative modifications in proteins such as histidine oxidation [34 -39] and tyrosine nitration [40,41], the development of techniques for ident...