TABLE 2.
Modified tryptic peptides of HSA reacted with diclofenac 1β-AG in vitro
HSA was incubated with synthetic diclofenac 1β-AG in phosphate buffer, pH 7.4, at 37°C for 16 hours. The peptides were characterized by LC-MS/MS (AB Sciex 5500 QTRAP). Glycated Lys162 was not detected consistently and was only ever seen at low signal strength (acylated Lys162 was never detected). Acylated Lys351 was not detected consistently and was only found at the 50:1 molar ratio. Acylated and glycated Lys436 were not detected consistently. Inconsistently observed adducts are shown as bold letters.
| Modified Lysinea | Tryptic Peptideb | Molar Ratio Diclofenac 1β-AG: HSA |
||||
|---|---|---|---|---|---|---|
| 0.01:1 | 0.1:1 | 1:1 | 10:1 | 50:1 | ||
| 137 | K*YLEIAR | — | — | — | G+T | G+T |
| 162 | YK*AAFTECCQAADK | — | — | — | G | G |
| 190 | LDELRDEGK*ASSAK | G | G | G+T | G+T | G+T |
| 195 | ASSAK*QR | — | — | G+T | G+T | G+T |
| 199 | LK*CASLQK | — | G | G+T | G+T | G+T |
| 351 | LAK*TYETTLEK | — | — | G | G | G+T |
| 432 | NLGK*VGSK | — | G | G | G+T | G+T |
| 436 | VGSK*CCK | — | — | — | G+T | G+T |
| 525 | K*QTALVELVK | — | — | G | G+T | G+T |
| 541 | ATK*EQLK | — | — | G+T | G+T | G+T |
G, complete glycation structure (diclofenac carboxyl and glucuronyl residues); T, diclofenac transacylation adduct.
Benoxaprofen AG modified Lys159 and Lys199 in vitro without reductive stabilization (Qiu et al., 1998). Tolmetin AG modified Lys195, 199, 525, and 541 in vitro without reductive stabilization (Ding et al., 1995). Lys159 and Lys199, respectively, were the principal adduction sites.
Asterisk indicates lysine modification site on the miscleaved peptide. The methodology of adduct identification is described under “Mass Spectrometric Characterization of Adducted Tryptic Peptides of HSA” in Materials and Methods. Cysteine residues of the recovered and reduced protein were carboxyamidomethylated before trypsin digestion.