High-resolution mass spectrometry analysis of protein oxidations and resultant loss of function

Abstract

MS, with or without pre-analysis peptide fractionation, can be used to decipher the residues on proteins where oxidative modifications caused by peroxynitrite, singlet oxygen or electrophilic lipids have occurred. Peroxynitrite nitrates tyrosine and tryptophan residues on the surface of actin. Singlet oxygen, formed by the interaction of UVA light with tryptophan, can oxidize neighbouring cysteine, histidine, methionine, tyrosine and tryptophan residues. Dose-response inactivation by 4HNE (4-hydroxynonenal) of hBAT (human bile acid CoA:amino acid N-acyltransferase) and CKBB (cytosolic brain isoform of creatine kinase) is associated with site-specific modifications. FT-ICR (Fourier-transform ion cyclotron resonance)-MS using nanoLC (nano-liquid chromatography)-ESI (electrospray ionization)-MS or direct-infusion ESI-MS with gas-phase fractionation identified 14 4HNE adducts on hBAT and 17 on CKBB respectively. At 4HNE concentrations in the physiological range, one member of the catalytic triad of hBAT (His362) was modified; for CKBB, although all four residues in the active site that were modifiable by 4HNE were ultimately modified, only one, Cys283, occurred at physiological concentrations of 4HNE. These results suggest that future in vivo studies should carefully assess the critical sites that are modified rather than using antibodies that do not distinguish between different modified sites.

Figure 1

ECD LTQ FT MS/MS spectrum of hBAT peptide ³³⁵AH*AEQAIGQLKR³⁴⁶. The spectrum contains a rich series of N-terminal c ions and C-terminal z ions. The c₂ ion has a m/z 364.234. The expected unmodified value for this ion is m/z 226.130. The difference is 138.104, the expected value for a Schiff base adduct, or a dehydrated Michael adduct. All the other c_n ions (n>2) () were increased by the same amount.

Figure 2

4HNE-induced inactivation of hBAT (□) and CK-BB (♦). Residual hBAT and CK-BB activities following incubation with 4HNE at varying concentrations are dose-dependent. Data are mean values of three independent experiments.

Figure 3

LTQ tandem mass spectrum of the triply oxidized αB-crystallin peptide ⁵⁷APSWFDTGLSEMR⁶⁹. The b₄ ion (m/z 474.2) is 218.1 bigger than the b₃ ion (m/z 256.1), showing that the Trp residue contains two oxygen atoms (186.1 + 2 × 16). Similarly, the y₂ ion (m/z 322.1) indicates that one oxygen atom has been added to the expected ion (m/z 306.1). Fragment ions that had undergone addition of oxygen to the Met residue (I) and/or the Trp residue (:) are marked.