Premium
Characterization of 4‐Hydroxy‐2‐Nonenal and 4‐Hydroxy‐2‐Hexenal modified proteins by Data Dependent Neutral Loss‐Triggered MS n
Author(s) -
Rauniyar Navin,
Stevens Stanley M,
Prokai Laszlo
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1025.3
Subject(s) - chemistry , mass spectrometry , fragmentation (computing) , adduct , electron capture dissociation , dissociation (chemistry) , collision induced dissociation , tandem mass spectrometry , peptide , ion , quadrupole ion trap , ion trap , chromatography , biochemistry , organic chemistry , computer science , operating system
End‐products of lipid peroxidation, 4‐hydroxy‐2‐nonenal (HNE) and 4‐hydroxy‐2‐hexenal (HHE), react with proteins to form covalent adducts. Mass spectrometric characterization of these adducts using a “bottom‐up” proteomic approach has been challenging because of the favored cleavage of the HNE/HHE modifications prior to backbone fragmentation of the peptide, preventing identification of the actual site of carbonylation due to the predominant neutral loss signal(s) observed in the MS/MS spectra. Our study involves characterization of HNE/HHE‐modified proteins using data dependent neutral loss‐triggered MSn with either collision induced dissociation (CID) or electron capture dissociation (ECD) via a hybrid linear ion trap FT‐ICR mass spectrometer. The general utility of NL‐driven MS3 acquisition using CID is evident from the enhanced characterization of protein‐HNE/HHE adducts observed by improving search algorithm scores to acceptable probability thresholds for identification. In the ECD method, peptide fragmentation is performed in the FTICR cell based on the neutral loss of HNE/HHE (156/114 Da) after MS/MS in the linear ion trap. The NL‐ECD method allowed for confident validation of protein modification by HNE/HHE and also identified several novel sites which were not definitively localized via NL‐CID. This research was supported by the grant AG025384 from the National Institutes of Health.