Fast Photochemical Oxidation Of Proteins Research Articles

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

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).

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

Protein radical labeling, like fast photochemical oxidation of proteins (FPOP), coupled to a top-down mass spectrometry (MS) analysis offers an alternative analytical method for probing protein structure or protein interaction with other biomolecules, for instance, proteins and DNA. However, with the increasing mass of studied analytes, the MS/MS spectra become complex and exhibit a low signal-to-noise ratio. Nevertheless, these difficulties may be overcome by protein isotope depletion. Thus, we aimed to use protein isotope depletion to analyze FPOP-oxidized samples by top-down MS analysis. For this purpose, we prepared isotopically natural (IN) and depleted (ID) forms of the FOXO4 DNA binding domain (FOXO4-DBD) and studied the protein-DNA interaction interface with double-stranded DNA, the insulin response element (IRE), after exposing the complex to hydroxyl radicals. As shown by comparing tandem mass spectra of natural and depleted proteins, the ID form increased the signal-to-noise ratio of useful fragment ions, thereby enhancing the sequence coverage by more than 19%. This improvement in the detection of fragment ions enabled us to detect 22 more oxidized residues in the ID samples than in the IN sample. Moreover, less common modifications were detected in the ID sample, including the formation of ketones and lysine carbonylation. Given the higher quality of ID top-down MSMS data set, these results provide more detailed information on the complex formation between transcription factors and DNA-response elements. Therefore, our study highlights the benefits of isotopic depletion for quantitative top-down proteomics. Data are available via ProteomeXchange with the identifier PXD044447.

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

Fast Photochemical Oxidation of Proteins (FPOP) is a promising technique for studying protein structure and dynamics. The quality of insight provided by FPOP depends on the reliability of the determination of the modification site. This study investigates the performance of two search engines, Mascot and PEAKS, for the data processing of FPOP analyses. Comparison of Mascot and PEAKS of the hemoglobin--haptoglobin Bruker timsTOF data set (PXD021621) revealed greater consistency in the Mascot identification of modified peptides, with around 26% of the IDs being mutual for all three replicates, compared to approximately 22% for PEAKS. The intersection between Mascot and PEAKS results revealed a limited number (31%) of shared modified peptides. Principal Component Analysis (PCA) using the peptide-spectrum match (PSM) score, site probability, and peptide intensity was applied to evaluate the results, and the analyses revealed distinct clusters of modified peptides. Mascot showed the ability to assess confident site determination, even with lower PSM scores. However, high PSM scores from PEAKS did not guarantee a reliable determination of the modification site. Fragmentation coverage of the modification position played a crucial role in Mascot assignments, while the AScore localizations from PEAKS often become ambiguous because the software employs MS/MS merging.

Efficient Analysis of Proteome-Wide FPOP Data by FragPipe.

Monitoring protein structure before and after environmental alterations (e.g., different cell states) can give insights into the role and function of proteins. Fast photochemical oxidation of proteins (FPOP) coupled with mass spectrometry (MS) allows for monitoring of structural rearrangements by exposing proteins to OH radicals that oxidize solvent-accessible residues, indicating protein regions undergoing movement. Some of the benefits of FPOP include high throughput and a lack of scrambling due to label irreversibility. However, the challenges of processing FPOP data have thus far limited its proteome-scale uses. Here, we present a computational workflow for fast and sensitive analysis of FPOP data sets. Our workflow, implemented as part of the FragPipe computational platform, combines the speed of the MSFragger search with a unique hybrid search method to restrict the large search space of FPOP modifications. Together, these features enable more than 10-fold faster FPOP searches that identify 150% more modified peptide spectra than previous methods. We hope this new workflow will increase the accessibility of FPOP to enable more protein structure and function relationships to be explored.

Application of in-cell fast photochemical oxidation of proteins (IC-FPOP) for the study of organoids.

Fast photochemical oxidation of proteins (FPOP) is a powerful, mass spectrometry (MS)-based, biophysical method used to probe protein structure, interactions, and conformations. FPOP was recently extended into cells (IC-FPOP) and can modify thousands of proteins in a single experiment, enabling proteome-wide structural biology. Although IC-FPOP can reveal critical structural information in 2D cell culture, the conditions do not emulate an in-vivo environment. Organoids are multicellular three-dimensional model systems that resemble the corresponding organ. They are the ideal 3D model system for evaluating drug penetration, drug efficacy, and chemoresistance since they mimic in vivo conditions. But the complexity of organoids makes them difficult for structural studies. Therefore, we have extended IC-FPOP into 3D bioprinted Huh-7 liver organoids. To obtain spatial resolution within the model, we integrated cryosectioning into the IC-FPOP workflow. This novel application of IC-FPOP revealed modifications in each section of the organoid, the top middle, and bottom. Pathway analysis revealed the interrogation of over 5 native pathways stemming from each region. Peptide level analysis revealed differences in the extent of modification for peptides identified in each region of the organoid, which confirms the acquisition of structural information. By coupling the organoid model with IC-FPOP we aim further validate its implementation for complex proteome-wide structural studies.

Development of Spheroid-FPOP: An In-Cell Protein Footprinting Method for 3D Tumor Spheroids.

Many cancer drugs fail at treating solid epithelial tumors with hypoxia and insufficient drug penetration thought to be contributing factors to the observed chemoresistance. Owing to this, it is imperative to evaluate potential cancer drugs in conditions as close to in vivo as possible, which is not always done. To address this, we developed a mass spectrometry-based protein footprinting method for exploring the impact of hypoxia on protein in 3D colorectal cancer cells. Our group has previously extended the protein footprinting method fast photochemical oxidation of proteins (FPOP) for live cell analysis (IC-FPOP); however, this is the first application of IC-FPOP in a 3D cancer model. In this study, we perform IC-FPOP on intact spheroids (Spheroid-FPOP) using a modified version of the static platform incubator with an XY movable stage (PIXY) FPOP platform. We detected modification in each of three spheroid layers, even the hypoxic core. Pathway analysis revealed protein modifications in over 10 distinct protein pathways, including some involved in protein ubiquitination; a process modulated in cancer pathologies. These results demonstrate the feasibility of Spheroid-FPOP to be utilized as a tool to interrogate protein interactions within a native tumor microenvironment.

Using mass spectrometry-based methods to understand amyloid formation and inhibition of alpha-synuclein and amyloid beta.

Amyloid fibrils, insoluble β-sheets structures that arise from protein misfolding, are associated with several neurodegenerative disorders. Many small molecules have been investigated to prevent amyloid fibrils from forming; however, there are currently no therapeutics to combat these diseases. Mass spectrometry (MS) is proving to be effective for studying the high order structure (HOS) of aggregating proteins and for determining structural changes accompanying protein-inhibitor interactions. When combined with native MS (nMS), gas-phase ion mobility, protein footprinting, and chemical cross-linking, MS can afford regional and sometimes amino acid spatial resolution of the aggregating protein. The spatial resolution is greater than typical low-resolution spectroscopic, calorimetric, and the traditional ThT fluorescence methods used in amyloid research today. High-resolution approaches can struggle when investigating protein aggregation, as the proteins exist as complex oligomeric mixtures of many sizes and several conformations or polymorphs. Thus, MS is positioned to complement both high- and low-resolution approaches to studying amyloid fibril formation and protein-inhibitor interactions. This review covers basics in MS paired with ion mobility, continuous hydrogen-deuterium exchange (continuous HDX), pulsed hydrogen-deuterium exchange (pulsed HDX), fast photochemical oxidation of proteins (FPOP) and other irreversible labeling methods, and chemical cross-linking. We then review the applications of these approaches to studying amyloid-prone proteins with a focus on amyloid beta and alpha-synuclein. Another focus is the determination of protein-inhibitor interactions. The expectation is that MS will bring new insights to amyloid formation and thereby play an important role to prevent their formation.

Fast photochemical oxidation of proteins coupled with mass spectrometry

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical footprinting approach whereby radicals, produced by UV laser photolysis of hydrogen peroxide, induce oxidation of amino acid side-chains. Mass Spectrometry (MS) is employed to locate and quantify the resulting irreversible, covalent oxidations to use as a surrogate for side-chain solvent accessibility. Modulation of oxidation levels under different conditions allows for the characterisation of protein conformation, dynamics and binding epitopes. FPOP has been applied to structurally diverse and biopharmaceutically relevant systems from small, monomeric aggregation-prone proteins to proteome-wide analysis of whole organisms. This review evaluates the current state of FPOP, the progress needed to address data analysis bottlenecks, particularly for residue-level analysis, and highlights significant developments of the FPOP platform that have enabled its versatility and complementarity to other structural biology techniques.

Top-Down Detection of Oxidative Protein Footprinting by Collision-Induced Dissociation, Electron-Transfer Dissociation, and Electron-Capture Dissociation.

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 a mixture of peptides from various subpopulations of proteins with varying degrees of photochemical oxidation. Here, we investigate the possibility to analyze a specifically selected population of only singly oxidized proteins. This requires utilization of more specific top-down mass spectrometry approaches. The key element of any top-down experiment is the selection of a suitable method of ion isolation, excitation, and fragmentation. Here, we employ and compare collision-induced dissociation, electron-transfer dissociation, and electron-capture dissociation combined with multi-continuous accumulation of selected ions. A singly oxidized subpopulation of FPOP-labeled ubiquitin was used to optimize the method. The top-down approach in FPOP is limited to smaller proteins, but its usefulness was demonstrated by using it to visualize structural changes induced by co-factor removal from the 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 good agreement, which indicates that monitoring a singly oxidized FPOP ion population by the top-down approach is a functional workflow for oxidative protein footprinting.

Utilization of Fast Photochemical Oxidation of Proteins and Both Bottom-up and Top-down Mass Spectrometry for Structural Characterization of a Transcription Factor-dsDNA Complex.

A combination of covalent labeling techniques and mass spectrometry (MS) is currently a progressive approach for deriving insights related to the mapping of protein surfaces or protein-ligand interactions. In this study, we mapped an interaction interface between the DNA binding domain (DBD) of FOXO4 protein and the DNA binding element (DAF16) using fast photochemical oxidation of proteins (FPOP). Residues involved in protein-DNA interaction were identified using the bottom-up approach. To confirm the findings and avoid a misinterpretation of the obtained data, caused by possible multiple radical oxidations leading to the protein surface alteration and oxidation of deeply buried amino acid residues, a top-down approach was employed for the first time in FPOP analysis. An isolation of singly oxidized ions enabled their gas-phase separation from multiply oxidized species followed by CID and ECD fragmentation. Application of both fragmentation techniques allowed generation of complementary fragment sets, out of which the regions shielded in the presence of DNA were deduced. The findings obtained by bottom-up and top-down approaches were highly consistent. Finally, FPOP results were compared with those of the HDX study of the FOXO4-DBD·DAF16 complex. No contradictions were found between the methods. Moreover, their combination provides complementary information related to the structure and dynamics of the protein-DNA complex. Data are available via ProteomeXchange with identifier PXD027624.

Optimization of a processing and analysis workflow for proteome-wide structural biology

Fast photochemical oxidation of proteins (FPOP) is a valuable, mass spectrometry (MS)-based, biophysical technique used to probe protein structure, interactions, and conformational states. FPOP has recently been extended to the study of intact cells (IC-FPOP) and has been shown to modify thousands of proteins in a single experiment for proteome-wide structural biology. Although IC-FPOP can reveal critical structural information, the post experimental process requires a multistep sample workflow to prepare for MS analysis and a lengthy data processing workflow post-MS analysis.

Development of a new automated platform for proteome-wide structural biology

In-cell fast photochemical oxidation of proteins (IC-FPOP) is a protein footprinting method that utilizes hydroxyl radicals to oxidatively modify the side chains of solvent accessible amino acids. Liquid chromatography coupled to mass spectrometry is used to both identify modified amino acids and quantify the levels of labeling. Owing to solvent accessibility changing upon binding or changes in conformations, IC-FPOP can be used to identify protein-ligand and protein-protein interaction sites and regions of conformational change.

The in-tandem application of qXL-MS and IC-FPOP to evaluate the inhibition of HSP90 by tanespimycin

Though healthy cells utilize the function of the 90 kDa heat shock protein (HSP90) to control cell growth, cell survival, and development -particularly in response to cellular stress- the activity of this protein is also beneficial to the growth and survival of cancer cells. Tanespimycin (17-AAG) is a well-characterized HSP90 inhibitor that has been shown to be cytotoxic to cancer cells. The complementary structural information gained from quantitative crosslinking with mass spectrometry (qXL-MS) and the In-Cell Fast Photochemical Oxidation of Proteins (IC-FPOP) was used to investigate the inhibition of HSP90 by 17-AAG in MCF-7 breast cancer cells.

Mass Spectrometry-Based Structural Proteomics for Metal Ion/Protein Binding Studies.

Metal ions are critical for the biological and physiological functions of many proteins. Mass spectrometry (MS)-based structural proteomics is an ever-growing field that has been adopted to study protein and metal ion interactions. Native MS offers information on metal binding and its stoichiometry. Footprinting approaches coupled with MS, including hydrogen/deuterium exchange (HDX), “fast photochemical oxidation of proteins” (FPOP) and targeted amino-acid labeling, identify binding sites and regions undergoing conformational changes. MS-based titration methods, including “protein–ligand interactions by mass spectrometry, titration and HD exchange” (PLIMSTEX) and “ligand titration, fast photochemical oxidation of proteins and mass spectrometry” (LITPOMS), afford binding stoichiometry, binding affinity, and binding order. These MS-based structural proteomics approaches, their applications to answer questions regarding metal ion protein interactions, their limitations, and recent and potential improvements are discussed here. This review serves as a demonstration of the capabilities of these tools and as an introduction to wider applications to solve other questions.

Hydroxyl radical footprinting analysis of a human haptoglobin-hemoglobin complex

Methods of structural mass spectrometry have become more popular to study protein structure and dynamics. Among them, fast photochemical oxidation of proteins (FPOP) has several advantages such as irreversibility of modifications and more facile determination of the site of modification with single residue resolution. In the present study, FPOP analysis was applied to study the hemoglobin (Hb) – haptoglobin (Hp) complex allowing identification of respective regions altered upon the complex formation. FPOP footprinting using a timsTOF Pro mass spectrometer revealed structural information for 84 and 76 residues in Hp and Hb, respectively, including statistically significant differences in the modification extent below 0.3%. The most affected residues upon complex formation were Met76 and Tyr140 in Hbα, and Tyr280 and Trp284 in Hpβ. The data allowed determination of amino acids directly involved in Hb – Hp interactions and those located outside of the interaction interface yet affected by the complex formation. Also, previously modeled interaction between Hb βTrp37 and Hp βPhe292 was not confirmed by our data. Data are available via ProteomeXchange with identifier PXD021621.

Carbocation Footprinting of Soluble and Transmembrane Proteins

Here, we introduce carbocations (R3C+) as laser-initiated footprinting reagents for proteins. We screened seven candidates and selected trifluomethoxy benzyl bromide (TFBB) as an effective precursor for the electrophilic trifluomethoxy benzyl carbocation (TFB+) under laser (248 nm) irradiation on the fast photochemical oxidation of proteins (FPOP) platform. Initial results demonstrate that this electrophilic cation reagent affords residue coverage of nucleophilic amino acids including H, W, M, and S. Further, the addition of TFB+ increases the hydrophobicity of the peptides so that separation of isomeric peptide products by reversed-phase LC is improved, suggesting opportunities for subresidue footprinting. Comparison of apo- and holo-myoglobin footprints shows that the TFB+ footprinting is sensitive to protein conformational change and solvent accessibility. Interestingly, because the TFB+ is amphiphilic, the reagent can potentially footprint membrane proteins as demonstrated for vitamin K epoxide reductase (VKOR) stabilized in a micelle. Not only does footprinting of the extra-membrane domain occur, but also some footprinting of the hydrophobic transmembrane domain is achieved owing to the interaction of TFB+ with the micelle. Carbocation precursors are stable and amenable for tailoring their properties and those of the incipient carbocation, enabling targeting their soluble or membrane-associated or embedded regions and distinguishing between the extra- and trans-membrane domains of membrane proteins.

Footprinting Mass Spectrometry of Membrane Proteins: Ferroportin Reconstituted in Saposin A Picodiscs.

Membrane proteins participate in a broad range of cellular processes and represent more than 60% of drug targets. One approach to their structural analyses is mass spectrometry (MS)-based footprinting including hydrogen/deuterium exchange (HDX), fast photochemical oxidation of proteins (FPOP), and residue-specific chemical modification. Studying membrane proteins usually requires their isolation from the native lipid environment, after which they often become unstable. To overcome this problem, we are pursuing a novel methodology of incorporating membrane proteins into saposin A picodiscs for MS footprinting. We apply different footprinting approaches to a model membrane protein, mouse ferroportin, in picodiscs and achieve high coverage that enables the analysis of the ferroportin structure. FPOP footprinting shows extensive labeling of the extramembrane regions of ferroportin and protection at its transmembrane regions, suggesting that the membrane folding of ferroportin is maintained throughout the labeling process. In contrast, an amphipathic reagent, N-ethylmaleimide (NEM), efficiently labels cysteine residues in both extramembrane and transmembrane regions, thereby affording complementary footprinting coverage. Finally, optimization of sample treatment gives a peptic-map of ferroportin in picodiscs with 92% sequence coverage, setting the stage for HDX. These results, taken together, show that picodiscs are a new platform broadly applicable to mass spectrometry studies of membrane proteins.

Evaluating the Sulfate Radical Anion as a New Reagent for In-Cell Fast Photochemical Oxidation of Proteins.

Fast photochemical oxidation of proteins (FPOP) has demonstrated the ability to inform on the higher order structure of proteins. Recent technological advances have extended FPOP to live cells (IC-FPOP) using multiple cell lines and in vivo (IV-FPOP) using C. elegans. These innovations allow proteins to be studied in their native cellular environment. Hydroxyl radicals are generated via the photoloysis of hydrogen peroxide. Hydrogen peroxide is a signaling molecule that can induce changes to some proteins in the cell limiting the proteins that can be studied by IC-FPOP. Here, we evaluate the sulfate radical anion as a footprinting label in IC-FPOP with sodium persulfate as the precursor. Our findings show a 1.5-fold increase in the number of modified proteins compared to IC-FPOP using hydroxyl radicals at the same precursor concentration demonstrating the amenability of this radical with IC-FPOP.

Platform Incubator with Movable XY Stage: A New Platform for Implementing In-Cell Fast Photochemical Oxidation of Proteins

Fast Photochemical Oxidation of proteins (FPOP) coupled with mass spectrometry (MS) has become an invaluable tool in structural proteomics to interrogate protein interactions, structure, and protein conformational dynamics as a function of solvent accessibility. In recent years, the scope of FPOP, a hydroxyl radical protein foot printing (HRPF) technique, has been expanded to protein labeling in live cell cultures, providing the means to study protein interactions in the convoluted cellular environment. In-cell protein modifications can provide insight into ligand induced structural changes or conformational changes accompanying protein complex formation, all within the cellular context. Protein footprinting has been accomplished employing a customary flow-based system and a 248 nm KrF excimer laser to yield hydroxyl radicals via photolysis of hydrogen peroxide, requiring 20 minutes of analysis for one cell sample.To facilitate time-resolved FPOP experiments, the use of a new 6-well plate-based IC-FPOP platform was pioneered. In the current system, a single laser pulse irradiates one entire well, which truncates the FPOP experimental time frame resulting in 20 seconds of analysis time, a 60-fold decrease. This greatly reduced analysis time makes it possible to research cellular mechanisms such as biochemical signaling cascades, protein folding, and differential experiments (i.e., drug-free vs. drug bound) in a time-dependent manner. This new instrumentation, entitled Platform Incubator with Movable XY Stage (PIXY), allows the user to perform cell culture and IC-FPOP directly on the optical bench using a platform incubator with temperature, CO2 and humidity control. The platform also includes a positioning stage, peristaltic pumps, and mirror optics for laser beam guidance. IC-FPOP conditions such as optics configuration, flow rates, transient transfections, and H2O2 concentration in PIXY have been optimized and peer-reviewed. Automation of all components of the system will reduce human manipulation and increase throughput.

Platform Incubator with Movable XY Stage: A New Platform for Implementing In-Cell Fast Photochemical Oxidation of Proteins.

Fast Photochemical Oxidation of proteins (FPOP) coupled with mass spectrometry (MS) has become an invaluable tool in structural proteomics to interrogate protein interactions, structure, and protein conformational dynamics as a function of solvent accessibility. In recent years, the scope of FPOP, a hydroxyl radical protein foot printing (HRPF) technique, has been expanded to protein labeling in live cell cultures, providing the means to study protein interactions in the convoluted cellular environment. In-cell protein modifications can provide insight into ligand induced structural changes or conformational changes accompanying protein complex formation, all within the cellular context. Protein footprinting has been accomplished employing a customary flow-based system and a 248 nm KrF excimer laser to yield hydroxyl radicals via photolysis of hydrogen peroxide, requiring 20 minutes of analysis for one cell sample.To facilitate time-resolved FPOP experiments, the use of a new 6-well plate-based IC-FPOP platform was pioneered. In the current system, a single laser pulse irradiates one entire well, which truncates the FPOP experimental time frame resulting in 20 seconds of analysis time, a 60-fold decrease. This greatly reduced analysis time makes it possible to research cellular mechanisms such as biochemical signaling cascades, protein folding, and differential experiments (i.e., drug-free vs. drug bound) in a time-dependent manner. This new instrumentation, entitled Platform Incubator with Movable XY Stage (PIXY), allows the user to perform cell culture and IC-FPOP directly on the optical bench using a platform incubator with temperature, CO2 and humidity control. The platform also includes a positioning stage, peristaltic pumps, and mirror optics for laser beam guidance. IC-FPOP conditions such as optics configuration, flow rates, transient transfections, and H2O2 concentration in PIXY have been optimized and peer-reviewed. Automation of all components of the system will reduce human manipulation and increase throughput.