Benefits of Ion Mobility Separation and Parallel Accumulation–Serial Fragmentation Technology on timsTOF Pro for the Needs of Fast Photochemical Oxidation of Protein Analysis

Abstract

Fast photochemical oxidation of proteins (FPOP) is a recently developed technique for studying protein folding, conformations, interactions, etc. In this method, hydroxyl radicals, usually generated by KrF laser photolysis of H 2 O 2 , are used for irreversible labeling of solvent-exposed side chains of amino acids. Mapping of the oxidized residues to the protein’s structure requires pinpointing of modifications using a bottom-up proteomic approach. In this work, a quadrupole time-of-flight (QTOF) mass spectrometer coupled with trapped ion mobility spectrometry (timsTOF Pro) was used for identification of oxidative modifications in a model protein. Multiple modifications on the same residues, including six modifications of histidine, were successfully resolved. Moreover, parallel accumulation–serial fragmentation (PASEF) technology allows successful sequencing of even minor populations of modified peptides. The data obtained indicate a clear improvement of the quality of the FPOP analysis from the viewpoint of the number of identified peptides bearing oxidative modifications and their precise localization. Data are available via ProteomeXchange with identifier PXD020509.

Chloride-Mediated Peroxide-Free Photochemical Oxidation of Proteins (PPOP) in Mass Spectrometry-Based Structural Analysis

“…According to the previous study, the reactivities of amino acid residues to • OH follow the sequence: Cys > Trp > Tyr > Met > Phe > His > Arg > Leu > Ile > other amino acids . Although • OH reacts with 16 or more types of residues, − the most common FPOP modifications of proteins in database searches are mono-oxidation for Met, Trp, Tyr, Phe, His, Leu, Ile, and Val residues and di-oxidation for Phe, Met, and Trp residues to simplify the data analysis. ,, To clearly demonstrate the residue types that can be oxidized in PPOP, we set oxidation variable modification (+15.99 Da) for 20 types of residues in the database searches of BSA-digested peptides as described in previous work . As a result, 19 types of oxidized residues were observed with reliable site localization (Supporting Information).…”

Safety and Efficacy of the Bordetella bronchiseptica Vaccine Combined with a Vegetable Oil Adjuvant and Multi-Omics Analysis of Its Potential Role in the Protective Response of Rabbits

“…Moreover, proteomic technology provides information regarding changes in protein expression and posttranslational regulation. The 4D label-free quantitative proteomic technique, which has a very high sensitivity and accurate quantification capability, can detect more low-abundance proteins than normal proteomic methods [ 53 , 54 , 55 ]. In this study, RNA-seq and 4D label-free proteomics were adopted to investigate the role of the E515 adjuvant in the E515- Bb vaccine in defense against Bb challenge.…”

Rhamnolipid Enhances the Nitrogen Fixation Activity of Azotobacter chroococcum by Influencing Lysine Succinylation

“…The 4D label-free quantitative proteomic approach is highly sensitive and capable of accurate quantification (Meier et al, 2018;Lesur et al, 2021;Loginov et al, 2021). This approach can detect more low-abundance proteins, but many lowabundance proteins were not identified with western blotting (Kurien and Scofield, 2015).…”