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).

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.

Antifungal triazoles affect key non-target metabolic pathways in Solanum lycopersicum L. plants.

Several 1,2,4-triazoles are widely used as systemic fungicides in agriculture because they inhibit fungal 14ɑ-demethylase. However, they can also act on many non-target plant enzymes, thereby affecting phytohormonal balance, free amino acid content, and adaptation to stress. In this study, tomato plants (Solanum lycopersicum L. var. 'Cherrola') were exposed to penconazole, tebuconazole, or their combination, either by foliar spraying or soil drenching, every week, as an ecotoxicological model. All triazole-exposed plants showed a higher content (1.7-8.8 ×) of total free amino acids than the control, especially free glutamine and asparagine were increased most likely in relation to the increase in active cytokinin metabolites 15 days after the first application. Conversely, the Trp content decreased in comparison with control (0.2-0.7 ×), suggesting depletion by auxin biosynthesis. Both triazole application methods slightly affected the antioxidant system (antioxidant enzyme activity, antioxidant capacity, and phenolic content) in tomato leaves. These results indicated that the tomato plants adapted to triazoles over time. Therefore, increasing the abscisic and chlorogenic acid content in triazole-exposed plants may promote resistance to abiotic stress.

Top-Down Proteoform Analysis by 2D MS with Quadrupolar Detection.

Two-dimensional mass spectrometry (2D MS) is a multiplexed tandem mass spectrometry method that does not rely on ion isolation to correlate the precursor and fragment ions. On a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS), 2D MS instead uses the modulation of precursor ion radii inside the ICR cell before fragmentation and yields 2D mass spectra that show the fragmentation patterns of all the analytes. In this study, we perform 2D MS for the first time with quadrupolar detection in a dynamically harmonized ICR cell. We discuss the advantages of quadrupolar detection in 2D MS and how we adapted existing data processing techniques for accurate frequency-to-mass conversion. We apply 2D MS with quadrupolar detection to the top-down analysis of covalently labeled ubiquitin with ECD fragmentation, and we develop a workflow for label-free relative quantification of biomolecule isoforms in 2D MS.

Triazoles as a Potential Threat to the Nutritional Quality of Tomato Fruits.

Triazole fungicides can threaten plants as abiotic stressors but can also positively affect plant defense by inducing priming. Thus, plant yield is also both protected and endangered by triazoles that may influence several metabolic pathways during maturation processes, such as the biosynthesis of saccharides or secondary metabolites. Here, Solanum lycopersicum L. plants were exposed to foliar and soil applications of penconazole, tebuconazole, or their combination, and their resulting effect on tomato fruits was followed. The exposure to the equimolar mixture of both triazoles influenced the representation of free proteinogenic amino acids, especially Gln, Glu, Gly, Ile, Lys, Ser and Pro, saccharide content, and led to a significant increase in the contents of total phenolics and flavonoids as well as positive stimulation of the non-enzymatic antioxidant system. Among the identified secondary metabolites, the most abundant was naringenin, followed by chlorogenic acid in tomato peel. In turn, all triazole-treated groups showed a significantly lower content of rosmarinic acid in comparison with the control. Foliar application of penconazole affected the fruit more than other single triazole applications, showing a significant decrease in antioxidant capacity, the total content of secondary metabolites, and the activities of total membrane-bound peroxidases and ascorbate peroxidase.

Casein as protein and hydrolysate: Biostimulant or nitrogen source for Nicotiana tabacum plants grown in vitro?

In contrast to inorganic nitrogen (N) assimilation, the role of organic N forms, such as proteins and peptides, as sources of N and their impact on plant metabolism remains unclear. Simultaneously, organic biostimulants are used as priming agents to improve plant defense response. Here, we analysed the metabolic response of tobacco plants grown in vitro with casein hydrolysate or protein. As the sole source of N, casein hydrolysate enabled tobacco growth, while protein casein was used only to a limited extent. Free amino acids were detected in the roots of tobacco plants grown with protein casein but not in the plants grown with no source of N. Combining hydrolysate with inorganic N had beneficial effects on growth, root N uptake and protein content. The metabolism of casein-supplemented plants shifted to aromatic (Trp), branched-chain (Ile, Leu, Val) and basic (Arg, His, Lys) amino acids, suggesting their preferential uptake and/or alterations in their metabolic pathways. Complementarily, proteomic analysis of tobacco roots identified peptidase C1A and peptidase S10 families as potential key players in casein degradation and response to N starvation. Moreover, amidases were significantly upregulated, most likely for their role in ammonia release and impact on auxin synthesis. In phytohormonal analysis, both forms of casein influenced phenylacetic acid and cytokinin contents, suggesting a root system response to scarce N availability. In turn, metabolomics highlighted the stimulation of some plant defense mechanisms under such growth conditions, that is, the high concentrations of secondary metabolites (e.g., ferulic acid) and heat shock proteins.

Effect of Agrimonia eupatoria L. and Origanum vulgare L. Leaf, Flower, Stem, and Root Extracts on the Survival of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the most antibiotic multi-resistant bacteria, causing chronic pulmonary disease and leading to respiratory failure and even mortality. Thus, there has been an ever-increasing search for novel and preferably natural antimicrobial compounds. Agrimonia eupatoria L. and Origanum vulgare L. shoots are commonly used as teas or alcoholic tinctures for their human health-promoting and antibacterial properties. Here, we explored the antimicrobial effects of all plant parts, i.e., leaf, flower, stem, and root extracts, prepared in water or in 60% ethanol, against P. aeruginosa. The impact of these extracts on bacterial survival was determined using a luminescent strain of P. aeruginosa, which emits light when alive. In addition, the antimicrobial effects were compared with the antioxidant properties and content of phenolic compounds of plant extracts. Ethanolic extracts of O. vulgare roots and flowers showed the highest antimicrobial activity, followed by A. eupatoria roots. In particular, chlorogenic acid, the ethanolic extract of O. vulgare roots contained high levels of protocatechuic acid, hesperidin, shikimic acid, rutin, quercetin, and morin. The synergistic effects of these phenolic compounds and flavonoids may play a key role in the antibacterial activity of teas and tinctures.

Counterintuitive structural and functional effects due to naturally occurring mutations targeting the active site of the disease-associated NQO1 enzyme.

Our knowledge on the genetic diversity of the human genome is exponentially growing. However, our capacity to establish genotype-phenotype correlations on a large scale requires a combination of detailed experimental and computational work. This is a remarkable task in human proteins which are typically multifunctional and structurally complex. In addition, mutations often prevent the determination of mutant high-resolution structures by X-ray crystallography. We have characterized here the effects of five mutations in the active site of the disease-associated NQO1 protein, which are found either in cancer cell lines or in massive exome sequencing analysis in human population. Using a combination of H/D exchange, rapid-flow enzyme kinetics, binding energetics and conformational stability, we show that mutations in both sets may cause counterintuitive functional effects that are explained well by their effects on local stability regarding different functional features. Importantly, mutations predicted to be highly deleterious (even those affecting the same protein residue) may cause mild to catastrophic effects on protein function. These functional effects are not well explained by current predictive bioinformatic tools and evolutionary models that account for site conservation and physicochemical changes upon mutation. Our study also reinforces the notion that naturally occurring mutations not identified as disease-associated can be highly deleterious. Our approach, combining protein biophysics and structural biology tools, is readily accessible to broadly increase our understanding of genotype-phenotype correlations and to improve predictive computational tools aimed at distinguishing disease-prone against neutral missense variants in the human genome.

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.

Structural basis for long-chain isoprenoid synthesis by cis-prenyltransferases.

Isoprenoids are synthesized by the prenyltransferase superfamily, which is subdivided according to the product stereoisomerism and length. In short- and medium-chain isoprenoids, product length correlates with active site volume. However, enzymes synthesizing long-chain products and rubber synthases fail to conform to this paradigm, because of an unexpectedly small active site. Here, we focused on the human cis-prenyltransferase complex (hcis-PT), residing at the endoplasmic reticulum membrane and playing a crucial role in protein glycosylation. Crystallographic investigation of hcis-PT along the reaction cycle revealed an outlet for the elongating product. Hydrogen-deuterium exchange mass spectrometry analysis showed that the hydrophobic active site core is flanked by dynamic regions consistent with separate inlet and outlet orifices. Last, using a fluorescence substrate analog, we show that product elongation and membrane association are closely correlated. Together, our results support direct membrane insertion of the elongating isoprenoid during catalysis, uncoupling active site volume from product length.

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.

Pythium oligandrum in plant protection and growth promotion: Secretion of hydrolytic enzymes, elicitors and tryptamine as auxin precursor.

Pythium is a genus of parasitic oomycetes which target plants and both nonvertebrate and vertebrate animals, including fish and mammalian species. However, several Pythium spp., such as P. oligandrum, function as mycoparasites of pathogenic fungi, bacteria, and oomycetes in soil and thus as advantageous biocontrol agents. This review primarily focuses on biochemical processes underlying their positive effects. For example, P. oligandrum degrades host cell wall polysaccharides using chitinases, cellulases, endo-ß-1,3-glucanases, and various exoglycosidases. Proteases from various classes also participate in the cell wall hydrolysis. All these processes can modify cell surface structures and help Pythium spp. compete for space and nutrition. Accordingly, enzyme secretion most likely plays a key role in plant root colonisation. Plant-P. oligandrum interactions, nevertheless, do not involve tissue injury but instead activate plant defence mechanisms, thereby strengthening future plant responses to pathogen attacks. Priming induces the phenylpropanoid and terpenoid pathways and thus synthesis of secondary metabolites, including lignin, for cell wall fortification and other metabolic adjustments. Such metabolic changes are mediated by elicitins, cell wall glycoproteins and oligandrins produced by P. oligandrum. As homologous proteins of ß-cinnamomin from Phytophthora cinnamomi with similar essential amino acids for sterol binding, oligandrins stand out for their structure, which they share with cell wall glycoproteins, albeit without the Ser-Thr-rich O-glycosylated domain for cell wall attachment. P. oligandrum also provides plant with tryptamine used for auxin synthesis, promoting plant growth. Overall, in addition to discussing plant metabolic and phytohormonal changes after P. oligandrum inoculation, we review data on P. oligandrum applications as researchers increasingly search for effective and environmentally friendly ways to protect crops. In this context, P. oligandrum emerges as a highly suitable biotechnological solution.

A single evolutionarily divergent mutation determines the different FAD-binding affinities of human and rat NQO1 due to site-specific phosphorylation.

The phosphomimetic mutation S82D in the cancer-associated, FAD-dependent human NADP(H):quinone oxidoreductase 1 (hNQO1) causes a decrease in flavin-adenine dinucleotide-binding affinity and intracellular stability. We test in this work whether the evolutionarily recent neutral mutation R80H in the vicinity of S82 may alter the strong functional effects of S82 phosphorylation through electrostatic interactions. We show using biophysical and bioinformatic analyses that the reverse mutation H80R prevents the effects of S82D phosphorylation on hNQO1 by modulating the local stability. Consistently, in rat NQO1 (rNQO1) which contains R80, the effects of phosphorylation were milder, resembling the behaviour found in hNQO1 when this residue was humanized in rNQO1 (by the R80H mutation). Thus, apparently neutral and evolutionarily divergent mutations may determine the functional response of mammalian orthologues towards phosphorylation.

How is the activity of shikimate dehydrogenase from the root of Petroselinum crispum (parsley) regulated and which side reactions are catalyzed?

Inhibitors of the shikimate pathway are widely used as herbicides, antibiotics, and anti-infectious drugs. However, the regulation of the shikimic pathway is complex, and little is known about the feedback regulation of the shikimate dehydrogenase (SDH, EC 1.1.1.25) in plants. Thus, the aim of this study was to elucidate the kinetic mechanism of SDH purified from the root of Petroselinum crispum (parsley), to determine all possible reaction products and to identify phenylpropanoid compounds that affect its activity. Our results showed that the bisubstrate reaction catalyzed by P. crispum SDH follows a sequential ordered mechanism, except for three dead-end complexes. The main and lateral reactions of SDH were monitored by mass spectrometry, thereby detecting protocatechuic acid as a byproduct. Gallic acid was formed non-enzymatically, whereas quinate was not detected. Several polyphenolic compounds inhibited SDH activity, especially tannic, caffeic and chlorogenic acids, with IC50 0.014 mM, 0.15 mM, and 0.19 mM, respectively. The number of hydroxyl groups influenced their inhibition effect on SDH, and p-coumaric, t-ferulic, sinapic, syringic and salicylic acids were less effective SDH inhibitors. Nevertheless, one branch of the phenylpropanoid pathway may affect SDH activity through feedback regulation.

LinX: A Software Tool for Uncommon Cross-Linking Chemistry.

Chemical cross-linking mass spectrometry has become a popular tool in structural biology. Although several algorithms exist that efficiently analyze data-dependent mass spectrometric data, the algorithm to identify and quantify intermolecular cross-links located at the interaction interface of homodimer molecules was missing. The algorithm in LinX utilizes high mass accuracy for ion identification. In contrast with standard data-dependent analysis, LinX enables the elucidation of cross-linked peptides originating from the interaction interface of homodimers labeled by 14N/15N, including their ratio or cross-links from protein-nucleic acid complexes. The software is written in Java language, and its source code and a detailed user's guide are freely available at https://github.com/KukackaZ/LinX or https://ms-utils.org/LinX. Data are accessible via the ProteomeXchange server with the data set identifier PXD023522.

Motif orientation matters: Structural characterization of TEAD1 recognition of genomic DNA.

TEAD transcription factors regulate gene expression through interactions with DNA and other proteins. They are crucial for the development of eukaryotic organisms and to control the expression of genes involved mostly in cell proliferation and differentiation; however, their deregulation can lead to tumorigenesis. To study the interactions of TEAD1 with M-CAT motifs and their inverted versions, the KD of each complex was determined, and H/D exchange, quantitative chemical cross-linking, molecular docking, and smFRET were utilized for structural characterization. ChIP-qPCR was employed to correlate the results with a cell line model. The results obtained showed that although the inverted motif has 10× higher KD, the same residues were affected by the presence of M-CAT in both orientations. Molecular docking and smFRET revealed that TEAD1 binds the inverted motif rotated 180°. In addition, the inverted motif was proven to be occupied by TEAD1 in Jurkat cells, suggesting that the low-affinity binding sites present in the human genome may possess biological relevance.

Studying Protein-DNA Interactions by Hydrogen/Deuterium Exchange Mass Spectrometry.

Protein hydrogen/deuterium exchange (HDX) coupled to mass spectrometry (MS) can be used to study interactions of proteins with various ligands, to describe the effects of mutations, or to reveal structural responses of proteins to different experimental conditions. It is often described as a method with virtually no limitations in terms of protein size or sample composition. While this is generally true, there are, however, ligands or buffer components that can significantly complicate the analysis. One such compound, that can make HDX-MS troublesome, is DNA. In this chapter, we will focus on the analysis of protein-DNA interactions, describe the detailed protocol, and point out ways to overcome the complications arising from the presence of DNA.

Three-Dimensional Printed Target Plates for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.

Three-dimensional printing (3D printing) is a fast-growing technology with high impact in industry, medicine, and the life sciences. Fused deposition modeling (FDM), which uses plastic filaments extruded through a heated nozzle, is the most common 3D printing technology for creation of objects. In this work, the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) target plates printed by FDM technology using conductive plastic material were evaluated for their detection capability of proteins and peptides. The 3D printed MALDI targets were validated by analysis of different types of bacteria and compared with commercially available MBT BioTargets. The results indicate that 3D printed MALDI targets are comparable to standard MBT BioTargets and stainless-steel targets and may be used for different MALDI-TOF MS applications. The 3D printing allows easy manufacturing of MALDI targets with different dimensions and spot geometry. Moreover, the 3D printed MALDI targets are disposable, cheap, and easy to produce. These features make them a suitable cost-effective alternative to conventional targets for any MALDI MS analysis.

The interaction of the mitochondrial protein importer TOMM34 with HSP70 is regulated by TOMM34 phosphorylation and binding to 14-3-3 adaptors.

Translocase of outer mitochondrial membrane 34 (TOMM34) orchestrates heat shock protein 70 (HSP70)/HSP90-mediated transport of mitochondrial precursor proteins. Here, using in vitro phosphorylation and refolding assays, analytical size-exclusion chromatography, and hydrogen/deuterium exchange MS, we found that TOMM34 associates with 14-3-3 proteins after its phosphorylation by protein kinase A (PKA). PKA preferentially targeted two serine residues in TOMM34: Ser93 and Ser160, located in the tetratricopeptide repeat 1 (TPR1) domain and the interdomain linker, respectively. Both of these residues were necessary for efficient 14-3-3 protein binding. We determined that phosphorylation-induced structural changes in TOMM34 are further augmented by binding to 14-3-3, leading to destabilization of TOMM34's secondary structure. We also observed that this interaction with 14-3-3 occludes the TOMM34 interaction interface with ATP-bound HSP70 dimers, which leaves them intact and thereby eliminates an inhibitory effect of TOMM34 on HSP70-mediated refolding in vitro In contrast, we noted that TOMM34 in complex with 14-3-3 could bind HSP90. Both TOMM34 and 14-3-3 participated in cytosolic precursor protein transport mediated by the coordinated activities of HSP70 and HSP90. Our results provide important insights into how PKA-mediated phosphorylation and 14-3-3 binding regulate the availability of TOMM34 for its interaction with HSP70.