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

## TL;DR

This paper introduces a new method using DIA data and spectral libraries to improve the analysis of protein structures through FPOP, offering better accuracy and reproducibility.

## Contribution

A novel workflow combining spectral libraries and DIA data is proposed to enhance FPOP data analysis accuracy and reproducibility.

## Key findings

- DIA and MS acquisition modes show better quantification reproducibility than DDA mode.
- Manually validated spectral libraries increase identification confidence and spatial resolution in FPOP analysis.
- The proposed workflow improves FPOP data processing by filtering nonrepresentative identifications.

## Abstract

Fast Photochemical
Oxidation of Proteins (FPOP) is a protein footprinting
method utilizing hydroxyl radicals to provide valuable information
on the solvent-accessible surface area. The extensive number of oxidative
modifications that are created by FPOP is both advantageous, leading
to great spatial resolution, and challenging, increasing the complexity
of data processing. The precise localization of the modification together
with the appropriate reproducibility is crucial to obtain relevant
structural information. In this paper, we propose a novel approach
combining validated spectral libraries together with utilizing DIA
data. First, the DDA data searched by FragPipe are subsequently validated
using Skyline software to form a spectral library. This library is
then matched against the DIA data to filter out nonrepresentative
IDs. In comparison with FPOP data processing using only a search engine
followed by generally applied filtration steps, the manually validated
spectral library offers higher confidence in identifications and increased
spatial resolution. Furthermore, the reproducibility of quantification
was compared for DIA, DDA, and MS-only acquisition modes on timsTOF
SCP. Comparison of coefficients of variation (CV) showed that the
DIA and MS acquisition modes exhibit significantly better reproducibility
in quantification (CV medians 0.1233 and 0.1494, respectively) compared
to the DDA mode (CV median 0.2104).