Transcription factor (TF) binding to DNA reshapes its backbone accessibility and modulate gene expression. However, protein–DNA interactions remain difficult to characterize with high resolution at the residue level. Here, we introduce Fast Photochemical Oxidation of Nucleic acids (FPON), a laser-driven hydroxyl-radical footprinting workflow coupled to liquid chromatography–high-resolution mass spectrometry (LC-MS) analysis. FPON quantitatively maps DNA accessibility and damage at sub-nucleotide resolution, revealing structural and interaction patterns induced by TF binding. Using the Forkhead box O4 DNA‑binding domain (FOXO4-DBD) bound to a double-stranded insulin response element (IRE), FPON generated structurally informative DNA fragments resolved and assigned by LC-MSn in a single experiment. Quantitative analysis identified site-specific protection patterns within the FOXO4 recognition motif. In the absence of a high-resolution structure, these site-specific protection patterns guided the construction of an in silico FOXO4–IRE complex. Complementary molecular dynamics simulations revealed a strong agreement between experimentally observed cleavage suppression and reduced solvent accessibility of DNA backbone hydrogens. Furthermore, FPON resolved multiple chemically distinct cleavage products otherwise indistinguishable in conventional gel-based footprinting approaches. Together, these results establish FPON as an unbiased, high-resolution strategy for mapping protein-DNA contacts readily applicable to more complex chromatin systems and dynamic transcriptional processes.

Heat shock cognate protein 70 (Hsc70) is a 71 kDa molecular chaperone belonging to the Hsp70 family of heat shock proteins. These proteins act as ATP-dependent molecular machines that assist protein folding under both physiological and stress conditions such as hypoxia, heat shock, and pH fluctuations. In addition to general chaperone functions, Hsc70 performs specialized roles, including uncoating clathrin-coated vesicles, facilitating protein transport into organelles, and targeting proteins for lysosomal degradation. Members of the Hsp70 family are known to form dimers and higher oligomers, but the structural organization and functional relevance of these assemblies remain poorly understood. Earlier studies also suggested that J-domain proteins (JDPs) can promote Hsp70 dimerization. In this study, we used chemical cross-linking, high-resolution Fourier transform mass spectrometry (FTMS), 15N isotopic labeling, and advanced data analysis to investigate the structural organization of Hsc70 dimers. Cross-link-derived distance restraints enabled structural modeling of Hsc70 monomers and dimers using AlphaLink2. Our results reveal distinct ATP- and ADP-state dimer conformations that coexist in equilibrium. In the presence of the cochaperone DnaJB1, we observed a shift in the dimer-monomer equilibrium, accompanied by enhanced ATP hydrolysis and formation of intermediate species. These findings demonstrate that the Hsc70 dimer population is structurally heterogeneous and depends on nucleotide state and cochaperone interactions.

This study evaluates the phytoremediation potential of Zea mays L. (DKC 3969) for removing psychotropic, antitussive, and anti-inflammatory pharmaceuticals from wastewater under sterile hydroponic conditions. Maize plants were exposed to buspirone (B), dextromethorphan (D), ibuprofen (I), paroxetine (P), sulpiride (S), and a combination of ibuprofen and dextromethorphan (I + D). Antidepressants B, P and S are increasingly used, but little is known about their fate in the environment and whether it will be possible to remove them in an ecologically friendly way. Results showed efficient uptake and translocation of B, D, and I, while P and S exhibited limited shoot translocation. Most drugs induced only mild oxidative stress, with S causing the strongest stress response but without affecting growth parameters, including elevated reactive oxygen species production and antioxidant activity. All pharmaceuticals elevated heat shock proteins HSP70 and HSP90 in maize leaves and roots, suggesting their role as biomarkers of xenobiotic-induced stress. Enhanced activity of glutathione-S-transferase and peroxidases, particularly in response to S and I + D, suggests initiation of biotransformation mechanisms. Overall, maize demonstrated resilience and effective pharmaceutical uptake, supporting its use as a phytoremediation strategy.

Proteolysis is a crucial step in both bottom-up and structural proteomics workflows, directly influencing peptide identification and sequence coverage in mass spectrometry-based analyses. While classical proteomics typically relies on highly specific enzymes with well-defined cleavage patterns, structural MS approaches such as hydrogen/deuterium exchange mass spectrometry (HDX-MS) often employ nonspecific or semispecific proteases, producing complex peptide mixtures that require more detailed digestion analysis. To address these needs and streamline the entire process, we developed DigDig, a standalone, Java-based software tool for evaluating and comparing proteolytic digestion across diverse experimental conditions. DigDig processes output files from common search engines and provides customizable visualizations of key digestion metrics, including sequence coverage, reproducibility, peptide redundancy, cleavage site preferences, and peptide length distributions. A distinguishing feature is its ability to detect and report repetitive peptide sequences, which are frequently missed by standard tools. We demonstrate its capabilities using data sets from both specific and nonspecific digestions, highlighting its utility in digestion quality control, protease characterization, and method development, particularly in HDX-MS workflows. DigDig is freely available at https://peterslab.org/DigDig/.

Abstract Background Phlebotomine sand flies (Diptera: Psychodidae) are important vectors of various pathogens, mainly Leishmania parasites. In the Old World, the most important genus in term of pathogens transmission is the genus Phlebotomus, which includes many proven or suspected vectors of several Leishmania species, while the genus Sergentomyia remains so far unproven as a vector of human pathogens. Algeria is one of the most affected countries by human leishmaniasis. Methods In the present study, an entomological survey was carried out in two provinces, Ghardaïa and Illizi, located in the north and central Sahara, respectively, where cases of human leishmaniasis are recorded. Our goal was to understand the role of the local sand fly species in the transmission of Leishmania parasites and to analyze their blood meal preferences. Collected sand flies were identified by a combination of morphological and molecular approaches that included DNA-barcoding and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) protein profiling. In addition, female blood meals were analyzed by peptide mass mapping using MALDI-TOF MS. Results In total, 640 sand fly specimens belonging to Phlebotomus and Sergentomyia genera were collected in the two provinces. Sergentomyia antennata and Se. fallax were most abundant species in Ghardaïa, and Ph. papatasi and Ph. alexandri in Illizi. In addition, a new sand fly species was described in Illizi named Sergentomyia (Sergentomyia) imihra n. sp. Blood meal analysis of the engorged females revealed various mammalian hosts, especially goats, but also humans for Phlebotomus papatasi and Ph. alexandri, suggesting that these vector species are opportunistic feeders. Conclusions Integrative approach that combined morphological analysis, sequencing of DNA markers, and protein profiling enabled the recognition and description of a new Sergentomyia species, raising the number of the Algerian sand fly fauna to 27 species. Further sand fly surveillance in the central Sahara is recommended to identify the thus-far unknown males of Se. imihra n. sp. Graphical 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).

High-risk human papillomaviruses (HPVs) cause various cancers. While type-specific prophylactic vaccines are available, additional anti-viral strategies are highly desirable. Initial HPV cell entry involves receptor-switching induced by structural capsid modifications. These modifications are initiated by interactions with cellular heparan sulphates (HS), however, their molecular nature and functional consequences remain elusive. Combining virological assays with hydrogen/deuterium exchange mass spectrometry, and atomic force microscopy, we investigate the effect of capsid-HS binding and structural activation. We show how HS-induced structural activation requires a minimal HS-chain length and simultaneous engagement of several binding sites by a single HS molecule. This engagement introduces a pincer-like force that stabilizes the capsid in a conformation with extended capsomer linkers. It results in capsid enlargement and softening, thereby likely facilitating L1 proteolytic cleavage and subsequent L2-externalization, as needed for cell entry. Our data supports the further devising of prophylactic strategies against HPV infections. The study shows that HPV activation requires a minimum glycan length, which enlarges and softens the virus. Using virological assays, AFM, and HDX-MS, the authors propose a model explaining HPV structural activation and its role in infection.

In proteomics, postproline cleaving enzymes (PPCEs), such as Aspergillus niger prolyl endopeptidase (AnPEP) and neprosin, complement proteolytic tools because proline is a stop site for many proteases. But while aiming at using AnPEP in online proteolysis, we found that this enzyme also displayed specificity to reduced cysteine. By LC-MS/MS, we systematically analyzed AnPEP sources and conditions that could affect this cleavage preference. Postcysteine cleavage was blocked by cysteine modifications, including disulfide bond formation, oxidation, and alkylation. The last modification explains why this activity has remained undetected so far. In the same experimental paradigm, neprosin mimicked this cleavage specificity. Based on these findings, PPCEs cleavage preferences should be redefined from post-Pro/Ala to post-Pro/Ala/Cys. Moreover, this evidence demands reconsidering PPCEs applications, whether cleaving Cys-rich proteins or assessing Cys status in proteins, and calls for revisiting the proposed enzymatic mechanism of these proteases.

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.

Several 1,2,4-triazoles are widely used as systemic fungicides in agriculture because they inhibit fungal 14alphademethylase. 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 x) 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 x), 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 triazoleexposed plants may promote resistance to abiotic stress.

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.

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.

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.

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.

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.

The cause of relapse in childhood acute lymphoblastic leukemia (ALL) is often associated with resistance to the standard chemotherapy treatment. The primary goal of this project was to elucidate the resistance mechanism of L-asparaginase (ASNase), one of the crucial drugs used in ALL therapy. The cytotoxic effect of ASNase relies on the depletion of extracellular asparagine (Asn) and glutamine, which is disastrous for leukemic cells since they have minimal activity of de novo synthesis of these amino acids that paradoxically have essential roles in leukemic cells' metabolism. We previously showed that ASNase caused metabolic reprogramming by which leukemic cells escaped the cytostatic effect of the treatment. In the present study, we investigated the role of the main aspects of the in vivo environment on the resistance mechanism of leukemic cells (BCP-ALL cell lines: NALM-6, REH, RS4-11 and SUP-B15 and primary ALL cells). By co-culturing them with mesenchymal stem cells (MSCs) and treating them with ASNase-pretreated culture media, we mimicked the bone marrow matrix and the in vivo half-life of the drug (1.28±0.35 days). The ASNase concentrations used in the ASNase-pretreated culture media were 0.04, 0.4 and 4 IU/mL. In concordance with previous results, we showed that leukemic cell survival was increased in the co-culture model compared to the "classical" in vitro treatment after five days using flow cytometry (NALM-6 - 0.04IU/mL: 17.37±2.8% p<0.0001, 0.4IU/mL: 18±2.8% p<0.0001, 4IU/mL: 25.87±2.3% p<0.0001; REH - 0.04IU/mL: 27.51±3.3% p<0.0001, 0.4IU/mL: 22.06±3.3% p<0.0001, 4IU/mL: 23.96±3.3% p<0.0001; RS4:11 - 0.04IU/mL: 50.07±2.9% p<0.0001, 0.4UI/mL: 60.14±2.9% p<0.0001, 4IU/mL: 34.50±3% p<0.0001; SUP-B15 - 0.04IU/mL: 13.90±2% p<0.0001, 0.4UI/mL: 19.55±2.3% p<0.0001, 4IU/mL: 5.72±2% p<0.05). While ASNase-mediated metabolic rewiring of leukemic cells persisted in both mono and co-culture: reduced glycolysis and increased fatty acid oxidation, the activity of mTOR-regulated biosynthetic pathways differed. The latter pathway was assessed by western blot quantification of the downstream targets of mTOR, S6 and CAD, which are protein and nucleotide synthesis mediators, respectively. In both cultures, the phosphorylated forms of S6 and CAD were inhibited after ASNase treatment (4IU/mL). However, the effect was significantly less profound in the co-culture model (REH: p-S6 (1.826 log (2) fold change, p=0.0043); p-CAD (2.385 log (2) fold change, p=0.0152), NALM-6: p-S6 (1.380 log (2) fold change, p=0.0106); p-CAD (0.78 log (2) fold change, p=n.s)). Similar changes in phospo-S6 were observed in primary BCP-ALL cells isolated from pediatric patients treated with ASNase. As shown by stable isotope tracing, asparagine synthesized de novo and released from MSCs compensated for asparagine depletion (after ASNase administration) and induced resistance of leukemic cells. Asparagine was sufficient to restore protein and nucleotide synthesis and partially rescued the viability of leukemic cells. In conclusion, the presence of MSCs sustains biosynthetic pathways, making leukemic cells more accessible to bioenergetic rewiring, which may counteract ASNase cytotoxicity. These findings present a potential therapeutical target for resistant patients. (Supported by GAČR GA20-27132S and GAUK 1262120)

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.

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.

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-β-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 β-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 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.