research articles Szapacs et al. Figure 5. LC-MS-MS of the T*H162LAPYSDELR tryptic peptide sequence from ApoA1 modiﬁed with KODA (A), with HNE (B) at His 162; of the QKL*H135ELQEK tryptic peptide sequence modiﬁed with 7-oxoheptanoic acid at His135 (C); and of the LEAL*K182ENGGAR tryptic peptide sequence modiﬁed with 8-oxooctanoic acid at Lys182 (D). or without mass shifts due to adducts. The program then maps adduct positions based on MS-MS fragmentation and calcu- lates probabilities of random matches.42 The ApoA1 search sequences used included miscleaved peptides that may result from lysine adduction by electrophiles. Our criteria for report- ing adducts included (1) presence of b-and/or y-ion series consistent with the adducted peptide sequence, (2) localization of adduct mass shifts by P-Mod to H or K residues capable of reacting with the electrophiles to form the indicated adducts, (3) presence of diagnostic water loss fragments formed from hydroxy-containing adducts (e.g., reduced Michael adducts). This analysis mapped sites of ApoA1 modiﬁcation and the detected mass shifts enabled identiﬁcation of the carboxylated electrophiles (Table 2). Annotated MS-MS spectra of these adducts are provided in Figure 5 and Figure S2 in Supporting Information. The identiﬁed residues include both lysine (K45, K106, K188, K195) and histidine residues (H135, H155, H162) and included both imine (Schiff base) and Michael adducts with carboxylated simple aldehydes, as well as carboxylated R, - 4242 Journal of Proteome Research Vol. 7, No. 10, 2008 unsaturated aldehydes (HODA and KODA). As noted above, the use of NaBH3CN as a reducing agent enabled us to distinguish KODA and HODA adducts. However, we note that KODA adducts corresponding in mass to Michael addition products may also be ketoamide adducts, which were recently described by Sayre and colleagues16 and which would not be distin- guished by our MS analyses. We note the formation of the apparently novel imine adducts of 7-oxoheptanoic acid, 8-oxooctanoic acid, and 9-oxononanoic acid in these analyses. The latter is formed by the Hock cleavage reaction that also generates HNE during linoleate oxidation.43 Formation of 8-oxooctanoic acid likely proceeds by fragmen- tation of the linoleate 9-hydroperoxide to give the 8-yl-octanoic acid. This radical would react with oxygen to give the 8-hy- droperoxyoctanoic acid which, upon dehydration, would give the 8-oxooctanoic acid compound. The mechanism for forma- tion of the 7-oxoheptanoic acid is not obvious. These analyses also identiﬁed some electrophile adducts derived from ω-terminal portion of the phospholipid-bound