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Metabolite-level regulation of enzyme activity is important for microbes to cope with environmental shifts. Knowledge of such regulations can also guide strain engineering to improve industrial phenotypes. Recently developed chemoproteomics workflows allow for genome-wide detection of metabolite-protein interactions that may regulate pathway activity. We applied limited proteolysis small molecule mapping (LiP-SMap) to identify and compare metabolite-protein interactions in the proteomes of two cyanobacteria and two lithoautotrophic bacteria that fix CO2 using the Calvin cycle. Clustering analysis of the hundreds of detected interactions showed that some metabolites interacted in a species-specific manner, such as interactions of glucose-6-phosphate in Cupriavidus necator and of glyoxylate in Synechocystis sp PCC 6803. These are interpreted in light of the different central carbon conversion pathways present. Metabolites interacting with the Calvin cycle enzymes fructose-1,6/sedoheptulose-1,7-bisphosphatase (F/SBPase) and transketolase were tested for effects on catalytic activity in vitro. The Calvin cycle intermediate glyceraldehyde-3-phosphate activated both Synechocystis and Cupriavidus F/SBPase, which suggests a feed-forward activation of the cycle in both photoautotrophs and chemolithoautotrophs. In contrast to the stimulating effect in reduced conditions, glyceraldehyde-3-phosphate inactivated the Synechocystis F/SBPase in oxidized conditions by accelerating protein aggregation. Thus, metabolite-level regulation of the Calvin cycle is more prevalent than previously appreciated and may act in addition to redox regulation., Not duplicate with DiVA 1608437QC 20230307

För att kunna livnära en växande population behöver avkastningen på skördar öka. En lösning på dettaär att optimera plantornas fotosyntes, vilket innefattar förbättrad koldioxidfixering. För att lyckas meddet krävs kunskap i hur reglering av nyckelproteiner i kloroplasten går till. Syftet med detta projekt är identifiera möjliga reglerande protein-metabolitinteraktioner i Arabidopsis thaliana. Målproteinerna ärde 11 enzymerna i Calvin-Benson-Basshamcykeln. Metaboliterna som testas är 3PGA, ATP, FBP, GAP, vilka är mellan produkter eller kofaktorer i cykeln; 2PG, som är en produkt av en konkurrerande reaktion i cykeln; och slutligen G6P, citrat och sackaros, vilka är centrala metaboliter i andra viktiga reaktioner i cellen. Före experimenten med Arabidopsis testades protokollen med spenat. Som ett första steg isolerades kloroplasterna från blad. När intakta kloroplaster verifierats extraherades proteinerna. Inter-aktioner mellan metaboliterna och proteinerna analyserades med en metod kallad limited proteolysis-small molecule mapping. Denna teknik, vilken kombinerar begränsad proteolys med masspektrometri, detekterade flertalet protein-metabolit interaktioner. I Arabidopsis uppvisade alla enzym förutom FB-Pase, PPE och TIM minst en interaktion. I spenat sågs interaktioner med FBA, GAPDH, PGK, PRK, RuBisCO, TIM och TK. Resultaten visar möjliga reglerande interaktioner, vilka skulle kunna användasför att identifiera flaskhalsar i kolfixeringen. Denna kunskap kan i sin tur utnyttjas för att öka flödet i Calvin-Benson-Basshamcykeln och därigenom förbättra växters koldioxidfixering., In order to feed a growing population, the crop yield needs to be increased. One way to do this is to optimise the photosynthetic activity in the plant, which includes improvement of carbon fixation. To succeed with this, knowledge of the regulation of key proteins in the chloroplast is required. The aim of this project is to identify possible regulatory protein-metabolite interactions in chloroplasts from Arabidopsis thaliana. The target proteins are the 11 enzymes of the Calvin-Benson-Bassham cycle. The metabolites of interest are 3PGA, ATP, FBP, GAP, which are intermediates or co-factors of the cycle;2PG, which is a product of a competing reaction in the cycle; and finally G6P, citrate and sucrose, which are central metabolites in other vital reactions in the cell. Before the experiments with Arabidopsis, spinach was used as a test organism to evaluate the proposed protocols. First, chloroplasts were isolatedfrom leaves. When the integrity of the chloroplasts had been validated, the proteins were extracted. Metabolic interactions with the extracted proteins were analyzed with limited proteolysis-small molecule mapping. This method, which combines limited proteolysis with mass spectrometry, detected severalprotein-metabolite interactions. In Arabidopsis, all enzymes except for FBPase, PPE and TIM had atleast one interaction. In spinach, interactions were seen with FBA, GAPDH, PGK, PRK, RuBisCO,TIM and TK. The results highlight potential regulatory events, which could be used to target bottlenecks in carbon fixation. This could provide a pathway to increase the flux in the Calvin-Benson-Bassham cycle, and thereby improve carbon fixation in plants.

Targeted proteomics is an attractive approach for the analysis of blood proteins. Here, we describe a novel analytical platform based on isotope-labeled recombinant protein standards stored in a chaotropic agent and subsequently dried down to allow storage at ambient temperature. This enables a straightforward protocol suitable for robotic workstations. Plasma samples to be analyzed are simply added to the dried pellet followed by enzymatic treatment and mass spectrometry analysis. Here, we show that this approach can be used to precisely (coefficient of variation <10%) determine the absolute concentrations in human plasma of hundred clinically relevant protein targets, spanning four orders of magnitude, using simultaneous analysis of 292 peptides. The use of this next-generation analytical platform for high-throughput clinical proteome profiling is discussed., QC 20220530

Metabolite-level regulation of enzyme activity is important for coping with environmental shifts. Recently developed proteomics methodologies allow for mapping of post-translational interactions, including metabolite-protein interactions, that may be relevant for quickly regulating pathway activity. While feedback and feedforward regulation in glycolysis has been investigated, there is relatively little study of metabolite-level regulation in the Calvin cycle, particularly in bacteria. Here, we applied limited proteolysis small molecule mapping (LiP-SMap) to identify metabolite-protein interactions in four Calvin-cycle harboring bacteria, including two cyanobacteria and two chemolithoautotrophs. We identified widespread protein interactions with the metabolites GAP, ATP, and AcCoA in all strains. Some species-specific interactions were also observed, such as sugar phosphates in Cupravidus necator and glyoxylate in Synechocystis sp. PCC 6803. We screened some metabolites with LiP interactions for their effects on kinetics of the enzymes F/SBPase and transketolase, two enzymatic steps of the Calvin cycle. For both Synechocystis and Cupriavidus F/SBPase, GAP showed an activating effect that may be part of feed-forward regulation in the Calvin cycle. While we verified multiple enzyme inhibitors on transketolase, the effect on kinetics was often small. Incorporation of F/SBPase and transketolase regulations into a kinetic metabolic model of Synechocystis central metabolism resulted in a general decreased stability of the network, and altered flux control coefficients of transketolase as well as other reactions. The LiP-SMap methodology is promising for uncovering new modes of metabolic regulation, but will benefit from improved peptide quantification and higher peptide coverage of enzymes, as known interactions are often not detected for low-coverage proteins. . Furthermore, not all LiP interactions appear to be relevant for catalysis, as 4/8 (transketolase) and 5/6 (F/S, QC 20211117

QC 20211125

Metabolite-level regulation of enzyme activity is important for coping with environmental shifts. Recently developed proteomics methodologies allow for mapping of post-translational interactions, including metabolite-protein interactions, that may be relevant for quickly regulating pathway activity. While feedback and feedforward regulation in glycolysis has been investigated, there is relatively little study of metabolite-level regulation in the Calvin cycle, particularly in bacteria. Here, we applied limited proteolysis small molecule mapping (LiP-SMap) to identify metabolite-protein interactions in four Calvin-cycle harboring bacteria, including two cyanobacteria and two chemolithoautotrophs. We identified widespread protein interactions with the metabolites GAP, ATP, and AcCoA in all strains. Some species-specific interactions were also observed, such as sugar phosphates in Cupravidus necator and glyoxylate in Synechocystis sp. PCC 6803. We screened some metabolites with LiP interactions for their effects on kinetics of the enzymes F/SBPase and transketolase, two enzymatic steps of the Calvin cycle. For both Synechocystis and Cupriavidus F/SBPase, GAP showed an activating effect that may be part of feed-forward regulation in the Calvin cycle. While we verified multiple enzyme inhibitors on transketolase, the effect on kinetics was often small. Incorporation of F/SBPase and transketolase regulations into a kinetic metabolic model of Synechocystis central metabolism resulted in a general decreased stability of the network, and altered flux control coefficients of transketolase as well as other reactions. The LiP-SMap methodology is promising for uncovering new modes of metabolic regulation, but will benefit from improved peptide quantification and higher peptide coverage of enzymes, as known interactions are often not detected for low-coverage proteins. . Furthermore, not all LiP interactions appear to be relevant for catalysis, as 4/8 (transketolase) and 5/6 (F/S, QC 20211117

The use of affinity reagents, such as antibodies, for studying specific molecules in complex backgrounds are some of the most powerful tools for researchers in molecular biology. However, all experiments performed using affinity reagents are directly affected by each reagent’s context-dependent ability to bind specifically to a target of interest. A growing issue with non-validated, or poorly validated affinity reagents, has been highlighted by the International Working Group for Antibody Validation (IWGAV). It has been suggested that antibodies should be evaluated in an application-specific manner since they can perform well in one application but fail to deliver reproducible results in another. One of the most commonly used antibody-based applications is the Western blot (WB) technology. When evaluating the result from a WB experiment, the initial measure used for determining whether or not the antibody binds the protein of interest is to determine the molecular weight of the protein detected by the antibody compared to a set of reference proteins. As WB relies on the SDS-PAGE for separating differently sized proteins, the comparison is actually based on protein migration during electrophoresis. It is, however, well known that the migration of a protein can differ significantly from how the reference proteins migrate. Here, we suggest a method for determining the actual migration patterns of proteins instead of relying on the theoretical molecular weight of the protein. Using this approach, called migration capture mass spectrometry (MS), a dataset containing the migration patterns of more than 39,000 protein products from more than 10,500 genes across eleven cell lines and tissues has been created. This migration capture MS approach has been validated using k-fold cross validation against 249 siRNA knockdown WBs showing that the method has a sensitivity of 96.4%, specificity of 87.4% and accuracy of 91.9%, which makes the dataset a useful resource that can facili, QC 20220927

Metabolite-level regulation of enzyme activity is important for coping with environmental shifts. Recently developed proteomics methodologies allow for mapping of post-translational interactions, including metabolite-protein interactions, that may be relevant for quickly regulating pathway activity. While feedback and feedforward regulation in glycolysis has been investigated, there is relatively little study of metabolite-level regulation in the Calvin cycle, particularly in bacteria. Here, we applied limited proteolysis small molecule mapping (LiP-SMap) to identify metabolite-protein interactions in four Calvin-cycle harboring bacteria, including two cyanobacteria and two chemolithoautotrophs. We identified widespread protein interactions with the metabolites GAP, ATP, and AcCoA in all strains. Some species-specific interactions were also observed, such as sugar phosphates in Cupravidus necator and glyoxylate in Synechocystis sp. PCC 6803. We screened some metabolites with LiP interactions for their effects on kinetics of the enzymes F/SBPase and transketolase, two enzymatic steps of the Calvin cycle. For both Synechocystis and Cupriavidus F/SBPase, GAP showed an activating effect that may be part of feed-forward regulation in the Calvin cycle. While we verified multiple enzyme inhibitors on transketolase, the effect on kinetics was often small. Incorporation of F/SBPase and transketolase regulations into a kinetic metabolic model of Synechocystis central metabolism resulted in a general decreased stability of the network, and altered flux control coefficients of transketolase as well as other reactions. The LiP-SMap methodology is promising for uncovering new modes of metabolic regulation, but will benefit from improved peptide quantification and higher peptide coverage of enzymes, as known interactions are often not detected for low-coverage proteins. . Furthermore, not all LiP interactions appear to be relevant for catalysis, as 4/8 (transketolase) and 5/6 (F/S, QC 20211117

Metabolite-level regulation of enzyme activity is important for coping with environmental shifts. Recently developed proteomics methodologies allow for mapping of post-translational interactions, including metabolite-protein interactions, that may be relevant for quickly regulating pathway activity. While feedback and feedforward regulation in glycolysis has been investigated, there is relatively little study of metabolite-level regulation in the Calvin cycle, particularly in bacteria. Here, we applied limited proteolysis small molecule mapping (LiP-SMap) to identify metabolite-protein interactions in four Calvin-cycle harboring bacteria, including two cyanobacteria and two chemolithoautotrophs. We identified widespread protein interactions with the metabolites GAP, ATP, and AcCoA in all strains. Some species-specific interactions were also observed, such as sugar phosphates in Cupravidus necator and glyoxylate in Synechocystis sp. PCC 6803. We screened some metabolites with LiP interactions for their effects on kinetics of the enzymes F/SBPase and transketolase, two enzymatic steps of the Calvin cycle. For both Synechocystis and Cupriavidus F/SBPase, GAP showed an activating effect that may be part of feed-forward regulation in the Calvin cycle. While we verified multiple enzyme inhibitors on transketolase, the effect on kinetics was often small. Incorporation of F/SBPase and transketolase regulations into a kinetic metabolic model of Synechocystis central metabolism resulted in a general decreased stability of the network, and altered flux control coefficients of transketolase as well as other reactions. The LiP-SMap methodology is promising for uncovering new modes of metabolic regulation, but will benefit from improved peptide quantification and higher peptide coverage of enzymes, as known interactions are often not detected for low-coverage proteins. . Furthermore, not all LiP interactions appear to be relevant for catalysis, as 4/8 (transketolase) and 5/6 (F/S, QC 20211117

Metabolite-level regulation of enzyme activity is important for coping with environmental shifts. Recently developed proteomics methodologies allow for mapping of post-translational interactions, including metabolite-protein interactions, that may be relevant for quickly regulating pathway activity. While feedback and feedforward regulation in glycolysis has been investigated, there is relatively little study of metabolite-level regulation in the Calvin cycle, particularly in bacteria. Here, we applied limited proteolysis small molecule mapping (LiP-SMap) to identify metabolite-protein interactions in four Calvin-cycle harboring bacteria, including two cyanobacteria and two chemolithoautotrophs. We identified widespread protein interactions with the metabolites GAP, ATP, and AcCoA in all strains. Some species-specific interactions were also observed, such as sugar phosphates in Cupravidus necator and glyoxylate in Synechocystis sp. PCC 6803. We screened some metabolites with LiP interactions for their effects on kinetics of the enzymes F/SBPase and transketolase, two enzymatic steps of the Calvin cycle. For both Synechocystis and Cupriavidus F/SBPase, GAP showed an activating effect that may be part of feed-forward regulation in the Calvin cycle. While we verified multiple enzyme inhibitors on transketolase, the effect on kinetics was often small. Incorporation of F/SBPase and transketolase regulations into a kinetic metabolic model of Synechocystis central metabolism resulted in a general decreased stability of the network, and altered flux control coefficients of transketolase as well as other reactions. The LiP-SMap methodology is promising for uncovering new modes of metabolic regulation, but will benefit from improved peptide quantification and higher peptide coverage of enzymes, as known interactions are often not detected for low-coverage proteins. . Furthermore, not all LiP interactions appear to be relevant for catalysis, as 4/8 (transketolase) and 5/6 (F/S, QC 20211117