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

ABSTRACT: We propose a novel approach for FPOP data analysis, utilizing DIA data. The HbHp protein complex was analyzed by FPOP and measured on timsToF SCP in DIA, DDA and MS modes. The IDs of modified peptides were quantified for each acquisition mode and the extent of modification was calculated on the level of peptides. The reproducibility was evaluated by coefficients of variation.This work was mainly financially supported by the Czech Science Foundation (22-27695S), the Technology Agency of the Czech Republic (ODEEP-EU TH86010001), the Ministry of Education, Youth and Sports of the Czech Republic grant PHOTOMACHINES - Photosynthetic cell redesign for high yields of therapeutic peptides (CZ.02.01.01/00/22_008/0004624) and the Academy of Sciences of the Czech Republic (RVO: 61388971).

INSTRUMENT(S): timsTOF SCP

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Marek Zakopcanik

LAB HEAD: Petr Novak

PROVIDER: PXD050132 | Pride | 2024-07-08

REPOSITORIES: pride

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Source:

			Action	DRS
	2024-01-16-decoys-DL_Hb_Hpt_all_cont_20220531.fasta.fas	Fasta
	DDA_HbHp1-1.zip	Other
	DDA_HbHp1-1_mid_samp_A1_1_1795.csv.gz	Csv
	DDA_HbHp1-1_mid_samp_A1_1_1795.mgf	Mgf
	DDA_HbHp1-1_mid_samp_A1_1_1796.csv.gz	Csv

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Project description:Fast photochemical oxidation of proteins (FPOP) footprinting is a structural mass spectrometry method that maps proteins by fast and irreversible chemical reactions. The position of oxidative modification reflects solvent accessibility and site reactivity and thus provides information about protein conformation, structural dynamics, and interactions. Bottom-up mass spectrometry is an established standard method to analyze FPOP samples. In the bottom-up approach, all forms of the protein are digested together by a protease of choice, which results in mixture of peptides from various subpopulations of proteins with varying degrees of photochemical oxidation. But, once the protein is already oxidized at least once, the spatial distribution of any consecutive oxidation no longer truly reflects initial protein solvent accessibility and residue reactivity, because the site preference of further oxidation may also be influenced by the changes caused by the prior oxidation. Thus, it would be interesting to obtain an assessment of the protein structure based on the FPOP data, by analyzing only singly oxidized protein populations. This requires utilization of more specific top-down mass spectrometry approaches. The key element of any top-down experiment is selection of a suitable method of ion isolation, excitation and fragmentation. Here we employ and compare collision-induced dissociation (CID), electron-transfer dissociation (ETD), and electron-capture dissociation (ECD) combined with multi continuous accumulation of selected ions (CASI). Singly oxidized subpopulation of FPOP labeled ubiquitin was used to optimize the method. The usefulness was then demonstrated further by using it to visualize structural changes induced by cofactor removal from holo/apo myoglobin system. The top-down data were compared with the literature and with the bottom-up data set obtained on the same samples. The top-down results were found to be in a good agreement, which indicates that monitoring singly FPOP oxidized ion population by top-down is a functional workflow for oxidative protein footprinting.

Dataset Information

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

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