Dataset Information

PASEF technology on timsTOF Pro improves quality of FPOP analysis

ABSTRACT: Fast photochemical oxidation of proteins (FPOP) is a modern technique for studying protein folding, conformations, interactions, etc. In this work, timsTOF Pro mass spectrometer coupled with trapped ion mobility spectrometry 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, PASEF technology allows successful sequencing even minor populations of modified peptides.

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Dmitry Loginov

LAB HEAD: Petr Novak

PROVIDER: PXD020509 | Pride | 2021-06-10

REPOSITORIES: Pride

ACCESS DATA

Publications

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

Loginov Dmitry S DS Fiala Jan J Chmelik Josef J Brechlin Peter P Kruppa Gary G Novak Petr P

ACS omega 20210408 15

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<sub>2</sub>O<sub>2</sub>, 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 t ...[more]

PMID: 34056188

Similar Datasets

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.

Project description:Tuberculosis (TB) still represents a major global health problem affecting over 10 million people worldwide. At present, there is an urgent need for defining prognostic parameters, differential diagnostic, and correlates of protection from the disease. The gold standard test for TB diagnosis is still smear microscopy, but insufficiently detects pulmonary disease, besides the necessity of having laboratory infrastructures and be time-consuming. Both LAM urine test and Xpert MTB/RIF have been major game changers in this context. However, the low sensitivity of the former and the considerable delay of sample delivery of the latter, make the improvement of the TB diagnostic landscape a priority. The development of rapid point-of-care (POC)-based testing approaches would represent an important breakthrough in TB diagnostics in view of these short-comings. Most forms of life produce extracellular vesicles (EVs) and since the first detection of bacterial EVs more than 60 years, subsequent studies have demonstrated their functional commonality despite differences in bacterial cell envelope architecture. We demonstrated that Mycobacterium tuberculosis (Mtb), the causative agent of TB, produces EVs in vitro and in vivo as part of a sophisticated mechanism to manipulate host cellular physiology and to evade the host immune system. In a previous serology study, Mtb EVs (MEVs) were used to investigate their potential role as biomarkers to discern between different forms of disease status. It was shown that the recognition of several MEV associated proteins could have diagnostic properties. In this study, we pursued to expand the capabilities of MEVs in the context of TB diagnostics by analyzing the composition of MEVs isolated from Mtb cultures submitted to iron starvation and, testing their immunogenicity against a new cohort of serum samples including TB+, LTBI and healthy donors. We found that MEVs differ in protein composition when Mtb is under host-related stress yet MEVs seem to carry antigens that could serve as bonafide markers for direct detection. In addition, TB serology revealed three new MEV antigens with great biomarker capacity. These results encourage investigating the feasibility of the development of a POC device based on selected MEV-associated proteins.

Dataset Information

PASEF technology on timsTOF Pro improves quality of FPOP analysis

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure