ap 3 a  (Jena Bioscience)

Journal: Nature Communications

Article Title: Chemical proteomic profiling reveals protein interactors of the alarmones diadenosine triphosphate and tetraphosphate

doi: 10.1038/s41467-021-26075-4

Figure Lengend Snippet: a Schematic workflow of PAL experiments. Probes were incubated with cell lysates and irradiated with UV light to initiate photo-crosslinking. Labeled proteins were affinity-purified using desthiobiotin (DTB) as affinity tag. Eluted fractions were digested and analyzed by LC-MS/MS and label-free quantification (LFQ). b Functionalized, non-hydrolysable Ap 3 A and Ap 4 A derivatives used as PAL probes (PALP). c Unlabeled, non-hydrolysable Ap 3 A and Ap 4 A derivatives and a control substance (con-1) lacking the Ap n A scaffold served as controls in PAL experiments.

Article Snippet: Ap 3 A and Ap 4 A (Jena Bioscience) were diluted in DcpS assay buffer (10 mM Tris∙HCl (pH 7.5), 200 mM KCl, 0.5 mM EDTA, 1.0 mM DTT) to a final concentration of 200 µM in 150 µL.

Techniques: Incubation, Irradiation, Labeling, Affinity Purification, Liquid Chromatography with Mass Spectroscopy

Journal: Nature Communications

Article Title: Chemical proteomic profiling reveals protein interactors of the alarmones diadenosine triphosphate and tetraphosphate

doi: 10.1038/s41467-021-26075-4

Figure Lengend Snippet: a Chemical structures of 13 C-labeled 13 C 10 -Ap 3 A ( n = 1) and 13 C 10 -Ap 4 A ( n = 2) used as internal standards. b Obtained Ap 3 A (green) and Ap 4 A (blue) levels of stressed and unstressed (control) HEK293T cells. For each condition 15 biological replicates were performed. Samples were measured in technical duplicates and averaged. Measurements with a signal to noise ratio below 7 were excluded. Data presented are mean ± standard error of the mean (SEM); n (control, Ap 3 A) = 9, n (control, Ap 4 A) = 7, n (MMC, Ap 3 A) = 12, n (MMC, Ap 4 A) = 11, n (H 2 O 2 , Ap 3 A) = 15, n (H 2 O 2 , Ap 4 A) = 9. Significant differences were determined by one-way ANOVA in combination with the Dunnett test (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001). Source data are provided as a Source Data file.

Article Snippet: Ap 3 A and Ap 4 A (Jena Bioscience) were diluted in DcpS assay buffer (10 mM Tris∙HCl (pH 7.5), 200 mM KCl, 0.5 mM EDTA, 1.0 mM DTT) to a final concentration of 200 µM in 150 µL.

Techniques: Labeling

Journal: Nature Communications

Article Title: Chemical proteomic profiling reveals protein interactors of the alarmones diadenosine triphosphate and tetraphosphate

doi: 10.1038/s41467-021-26075-4

Figure Lengend Snippet: a Western blot analysis with ExtrAvidin®-Peroxidase showing the enrichment of labeled proteins under optimized conditions using PALP-1 (left) and PALP-2 (right) as indicated after irradiation with UV light (365 nm) for 5 min. b As for a but usage of PALP-3. Structure of PALP-3 is shown. c Heat map representation ( Z -scores) of proteins that were quantified in the respective experiments in at least four out of six measurements and that passed a one-way ANOVA-based multiple-sample test for statistically significant enrichment, with a permutation-based false discovery rate (FDR) below or equal to 0.01 and S 0 value set to 0.2, followed up by two-sided post hoc Tukey’s HSD test (FDR ≤ 0.05) to filter for significant enrichment against all performed control experiments. d Venn diagram showing the distribution of identified proteins for Ap 3 A probes (data obtained with PALP-1 and PALP-3, green) and the Ap 4 A probe (data obtained with PALP-2, blue). e Extract of biological processes that are significantly overrepresented in proteins found with Ap 3 A-based probes (PALP-1 and PALP-3, green) and the Ap 4 A-based probe (PALP-2, blue) compared to the global human proteome (gray) according to GO analysis using a one-sided hypergeometric test performed with the Cytoscape BiNGO app (Supplementary Data ). Global frequency represents the number of genes annotated to a GO term in the entire human proteome, while sample frequency represents the number of genes annotated to that GO term in the protein list obtained by the presented PAL approaches. Source data are provided as a Source Data file.

Article Snippet: Ap 3 A and Ap 4 A (Jena Bioscience) were diluted in DcpS assay buffer (10 mM Tris∙HCl (pH 7.5), 200 mM KCl, 0.5 mM EDTA, 1.0 mM DTT) to a final concentration of 200 µM in 150 µL.

Techniques: Western Blot, Labeling, Irradiation

Journal: Nature Communications

Article Title: Chemical proteomic profiling reveals protein interactors of the alarmones diadenosine triphosphate and tetraphosphate

doi: 10.1038/s41467-021-26075-4

Figure Lengend Snippet: a Extract of ANOVA-heatmap ( Z -scores) for DcpS under different PAL conditions. b DcpS catalyzed cleavage of m 7 Gp 3 G. c Excerpt of the HPLC profiles for the hydrolysis of m 7 Gp 3 G (peak c) catalyzed by DcpS. The initial substrate concentration was 20 µM and the reactions were carried out in the absence (gray) or presence of Ap 3 A (green) and Ap 4 A (blue) (200 µM) at an enzyme concentration of 100 n M . Absorbance was measured at 260 nm (arb. units, arbitrary units). Formation of GDP (peak a) can be observed for all reactions. For Ap 3 A (peak f), ADP (peak b) and AMP (peak d) were identified as hydrolysis products. By analogy, AMP (peak d) and ATP (peak e) were identified as the cleavage products of Ap 4 A (peak g). The chromatographic peaks were identified by comparison with the retention times of reference samples and by subsequent MS analysis. d Quantification of the cleavage product GDP at different time points, applying adenosine as internal standard (IS). Data presented are mean ± SEM, n = 3 biologically independent experiments. Source data are provided as a Source Data file.

Article Snippet: Ap 3 A and Ap 4 A (Jena Bioscience) were diluted in DcpS assay buffer (10 mM Tris∙HCl (pH 7.5), 200 mM KCl, 0.5 mM EDTA, 1.0 mM DTT) to a final concentration of 200 µM in 150 µL.

Techniques: Concentration Assay

Journal: Nature Communications

Article Title: Chemical proteomic profiling reveals protein interactors of the alarmones diadenosine triphosphate and tetraphosphate

doi: 10.1038/s41467-021-26075-4

Figure Lengend Snippet: a Extract of ANOVA-heatmap ( Z -scores) for UBA1 under different PAL conditions. b ATP-dependent Ub activation and transthioesterification reactions catalyzed by UBA1. c Detection of Ub transfer from UBA1 to UbcH7 in the presence of Ap 3 A, Ap 4 A or ATP, analyzed by non-reducing SDS-PAGE (15%) followed by Coomassie blue staining. d The nucleotides were incubated with SAP before performing the transthioesterification reaction to remove possible contaminants of ATP in the Ap 3 A and Ap 4 A samples. The experiments were performed twice with matching results. Source data are provided as a Source Data file.

Article Snippet: Ap 3 A and Ap 4 A (Jena Bioscience) were diluted in DcpS assay buffer (10 mM Tris∙HCl (pH 7.5), 200 mM KCl, 0.5 mM EDTA, 1.0 mM DTT) to a final concentration of 200 µM in 150 µL.

Techniques: Activation Assay, SDS Page, Staining, Incubation

Journal: Nature Communications

Article Title: Chemical proteomic profiling reveals protein interactors of the alarmones diadenosine triphosphate and tetraphosphate

doi: 10.1038/s41467-021-26075-4

Figure Lengend Snippet: a Extract of ANOVA-heatmap ( Z -scores) for LDHA and PGK1 under different PAL conditions. b Chemical structure of F-Ap 3 A. c FP-based quantification of binding between LDHA (orange) or PGK1 (green) to F-Ap 3 A. Increasing concentrations of protein were incubated with F-Ap 3 A (25 n M ) for 15 min on ice and 30 min at 30 °C before measurement. FP values were determined and plotted against the protein concentrations. Data presented are mean ± SD, n = 3 technical replicates. Source data are provided as a Source Data file.

Article Snippet: Ap 3 A and Ap 4 A (Jena Bioscience) were diluted in DcpS assay buffer (10 mM Tris∙HCl (pH 7.5), 200 mM KCl, 0.5 mM EDTA, 1.0 mM DTT) to a final concentration of 200 µM in 150 µL.

Techniques: Binding Assay, Incubation