Fast photochemical oxidation of protein: DIA workflow and the impact of the peptide identification framework

Methods of structural mass spectrometry facilitate structural studies of protein folding and interaction. Fast photochemical oxidation of protein (FPOP) utilizes •OH for the protein labelling occurring on a microsecond timescale and possess a broad reactivity. Accurate interpretation of MSMS spectra is a key point for the FPOP analysis. This task is solved by using different search engines which differ in technique for spectrum identification and scoring systems, providing unique identifications in addition to set of mutual identifications. In this study we compared the reliability of peptide identifications by two different search engines and evaluated the perspectives of using spectral libraries in combination with DIA acquisition to increase the precision of FPOP modification assignment.

A comparison of the search engines Mascot and PEAKS X+ was conducted using the data set PXD021621 (ProteomeXchange). The results demonstrated that the choice of search engine significantly affects the assignment of FPOP modifications to detected peptides, with only 31% of identifications common to both engines. PCA revealed distinct clusters of modified peptides based on score, site probabilities, and intensities. Mascot showed more confident site determination, with a clear correlation between fragmentation spectrum quality and assigned site confidence. Consequently, Mascot required less manual validation due to the majority of identifications belonging to a reliable top cluster. In contrast, PEAKS ID clustering lacked a clear connection to identification reliability, necessitating extensive manual validation.

We propose a novel approach that combines validated spectral libraries with DIA data. First, DDA data searched by FragPipe are validated using Skyline software to form a spectral library. This library is then matched against the DIA data to filter out non-representative IDs. Out of 421 accepted identifications, 346 contained FPOP modifications, primarily oxidations and dioxidations. Comparing the manually validated library with Mascot search results revealed that the library offers a wider range of unique modifications, increased spatial resolution, and better coverage of biologically relevant areas of the protein complex. For the first time, a DIA workflow was used for FPOP data with a manually validated spectral library.

This work was supported by Czech Scientific Foundation (22-27695S), Technology Agency of the Czech Republic (ODEEP-TH86010001) and by Programme Johannes Amos Comenius Photomachines (CZ.02.01.01/00/22_008/0004624).