Charge Derivatized Amino Acids Facilitate Model Studies on Protein Side-Chain Modifications by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

摘要

The α-amino groups of histidine and lysine were derivatized with p-carboxylbenzyltriphenylphosphonium to form the pseudo dipeptides, PHis and PLys, which can be sensitively detected by MALDITOF mass spectrometry due to the fixed positive charge of the phosphonium group. Detection limits of PHis and PLys by MALDITOF mass spectrometry were both 30 fmol with a signal-to-noise ratio of 5:1. These pseudo dipeptides were excellent surrogates for His or Lys-containing peptides in model reactions mimicking proteins with reactive electrophiles, prominently those generated by peroxidation of polyunsaturated fatty acids including 4-hydroxy-2(E)-nonenal (HNE), 4-oxo-2(E)-nonenal (ONE), 2(E)-octenal, and 2(E)-heptenal. An air saturated solution of linoleic acid (d0:d5 = 1:1) was incubated in the presence of Fe(II) and ascorbate with these two pseudo dipeptides, and the reaction products were characterized by MALDITOFMS and LCESIMS. By using PHis and PLys, the previously reported ONE-derived His-furan adduct was detected along with evidence for a cyclic α,β-unsaturated ketone. A dimer formed from ONE was found to react with PHis through Michael addition. Alkenals were found to form two novel adducts with PLys. 2(E)-Octenoic acid–His Michael adduct and N^ε-pentanoyllysine were identified as potential protein side-chain adducts modified by products of linoleic acid peroxidation. In addition, when PHis or PLys and AcHis or BocLys were exposed to the linoleic acid peroxidation, an epoxy-keto-ocatadecenoic acid mediated His–His cross-link was detected, along with a His–ONE/9,12-dioxo-10-dodecenoic acid–Lys derived pyrrole cross-link was observed.