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 H2O2, 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.

Bruker Daltonik GmbH Fahrenheitstraße 4 28359 Bremen Germany

Bruker s r o Prazakova 60 619 00 Brno Czech Republic

Faculty of Science Charles University Hlavova 8 128 20 Prague Czech Republic

Institute of Microbiology The Czech Academy of Sciences Videnska 1083 14220 Prague Czech Republic

Orekhovich Institute of Biomedical Chemistry Pogodinskaja str 10 119191 Moscow Russia

ACS Omega. 2021 May 28;6(22):14726. doi: 10.1021/acsomega.1c02648. PubMed

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Data-Independent Acquisition Represents a Promising Alternative for Fast Photochemical Oxidation of Proteins (FPOP) Samples Analysis

Quantifying the Impact of the Peptide Identification Framework on the Results of Fast Photochemical Oxidation of Protein Analysis

Isotopic Depletion Increases the Spatial Resolution of FPOP Top-Down Mass Spectrometry Analysis