Correct identification of oxidized histidine residues using electron‐transfer dissociation

Abstract

Oxidative modification to the side chain of histidine can noticeably change the collision-induced dissociation (CID) pathways of peptides containing this oxidized residue. In cases where an oxidized peptide consists of two or more isomers differing only in the site of modification, oxidation to histidine usually causes the other oxidized sites to be mis-assigned in CID spectra. These spectral mis-assignments can sometimes be avoided by using multiple stages of MS/MS (MS n ) or via specially-optimized liquid chromatographic separation conditions. In this manuscript, we demonstrate that these mis-assignments can be more readily and easily avoided by using electrontransfer dissociation (ETD) to dissociate the oxidized peptides. Furthermore, we find that the relative insensitivity of ETD to side chain chemistry allows the extent of oxidative modification to be determined readily for peptide isomers having more than one site of oxidation. The current results along with previous studies of oxidized peptides suggest that ETD is probably a better technique than CID for obtaining correct sequence and modification information for oxidized peptides.

Quantitative Protein Topography Analysis and High-Resolution Structure Prediction Using Hydroxyl Radical Labeling and Tandem-Ion Mass Spectrometry (MS)*

“…Results-There can be variation in the fragmentation chemistry of oxidized versus unoxidized peptide isomers during the collision-induced dissociation, which can potentially confound the results in absolute terms (35,36,43,44). Such variations can also lead to negative bias, some example cases are shown by negative values of the RC (Table I).…”

Supercharging by m-NBA Improves ETD-Based Quantification of Hydroxyl Radical Protein Footprinting

“…Initial uses of HRPF were limited in spatial resolution to the size of a proteolytic peptide, as the amount of oxidation of any individual amino acid within the peptide could not be accurately quantified by CID [8-10]. As sub-microsecond HRPF technologies such as Fast Photochemical Oxidation of Proteins (FPOP) [3] and pulsed electron beam radiolysis [11] began to allow for heavier oxidation of proteins, the need to quantitate isomeric peptide oxidation products became even more pronounced.…”

Electron Transfer Dissociation of Modified Peptides and Proteins

“…This occurs because the dissociation chemistry of each oxidized isomer can be very different during CID, which gives rise to complicated spectra that are easy to mis-interpret [49,50]. This problem can be overcome if specialized LC separation conditions are employed to fully separate each oxidized isomer, but these LC conditions must be optimized for each isomer mixture, something that is prohibitive when multiple samples must be analyzed.…”

“…This problem can be overcome if specialized LC separation conditions are employed to fully separate each oxidized isomer, but these LC conditions must be optimized for each isomer mixture, something that is prohibitive when multiple samples must be analyzed. Again, because ETD is less sensitive to side chain chemistry, this dissociation technique can be used to accurately identify oxidation sites for peptide isomers upon dissociating them simultaneously [50]. It has been found that oxidized peptide isomers tend to dissociate in similar manners during ETD, thereby avoiding the mis-interpretations that are common in CID of such isomers.…”