Charge-derivatized amino acids facilitate model studies on protein side-chain modifications by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The alpha-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 matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) due to the fixed positive charge of the phosphonium group. Detection limits of PHis and PLys by MALDI-TOFMS 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 MALDI-TOFMS and liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). By using PHis and PLys, the previously reported ONE-derived His-furan adduct was detected along with evidence for a cyclic alpha,beta-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(epsilon)-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 the observation of a His-ONE/9,12-dioxo-10-dodecenoic acid-Lys derived pyrrole cross-link.