phosphopeptide enrichment  (Tymora Analytical Operations LLC)

Journal: Frontiers in Cell and Developmental Biology

Article Title: Inducible degradation-coupled phosphoproteomics identifies PP2A Rts1 as a novel eisosome regulator

doi: 10.3389/fcell.2024.1451027

Figure Lengend Snippet: Optimization of AID phosphoproteomics method. (A) Final AID-phosphoproteomics workflow. Parent culture expresses 1) target of interest tagged with AID at its natural locus and 2) OsTIR1 from the ADH1 promoter. The phosphopeptide enrichment step includes SCX, followed by PolyMAC. IAA treatment is for 35 min. See Methods for full details. (B) Effects of bulk SCX pre-enrichment of phosphopeptide pool in reducing background of unmodified peptides. (C) Representative effect of SCX pre-enrichment on the final fraction of phosphopeptides after PolyMAC step. Identified phosphopeptides and unmodified peptides from LC-MS/MS were used to create the bar graph. (D) Representative distribution of phosphopeptides and unmodified peptides identified by LC-MS/MS analysis in the C18 high pH reverse phase fractions generated by elution with the indicated concentrations of acetonitrile. Data shown in (B-D) are from single analyses of final conditions after the optimization process using strain YKA1233.

Article Snippet: Phosphopeptides were further enriched using PolyMAC ( ) phosphopeptide kit (Tymora Analytical) as described by the manufacturer with the following adjustments.

Techniques: Liquid Chromatography with Mass Spectroscopy, Generated

Journal: bioRxiv

Article Title: LRRK2 mediates haloperidol-induced changes in indirect pathway striatal projection neurons

doi: 10.1101/2024.06.06.597594

Figure Lengend Snippet: A. Schematic of experimental procedures. WT and GS mutant mice were treated with haloperidol or vehicle, and striatal samples were harvested after 1 hour. LC-MS/MS analyses for total proteins and phosphopeptides were conducted on the same striatal samples. N=3 mice/group. Parts of the schematic were created with BioRender.com. B. Volcano plot comparing the phosphopeptides between haloperidol and vehicle-treated WT mice. Phosphopeptides that are significantly differentially regulated (|Log2FC| > 2 and unadjusted p-value < 0.05 by multiple unpaired t-tests) are colored red and blue for up- and down-regulated, respectively. C. Results of Gene Set Enrichment Analysis (GSEA) of the KEGG gene set for genes with at least one differentially regulated phosphopeptide in WT haloperidol vs vehicle-treated comparison. Pathways displayed are significantly differently regulated (adjusted p-value < 0.05 by Fisher’s test). The length of bars reflects the number of genes in the pathway whose phosphostate is differentially regulated; bars are shaded by adjusted p-value. Highlighted pathways are related to established LRRK2 functions. D. Diagram of genetic and pharmacological conditions for groups analyzed in E. Created with BioRender.com. E. Heatmap of effect size (Log2FC) of either haloperidol treatment or LRRK2-GS for all differentially abundant phosphopeptides in the WT haloperidol-treated vs vehicle-treated comparison (|Log2FC| > 2 and unadjusted p-value < 0.05 by multiple unpaired t-tests). Each bar represents a phosphopeptide. Color reflects Log2FC difference from WT saline condition. F. Correlation plot for E, comparing GS vehicle/WT vehicle to WT haloperidol-vehicle effect size. All detected phosphopeptides were mapped, and phosphopeptides significantly altered between WT haloperidol vs. vehicle are highlighted. Only highlighted values are used for correlation calculation. Blue line represents the line of best fit.

Article Snippet: The samples were dried completely in a vacuum centrifuge and used for phosphopeptide enrichment using PolyMAC Phosphopeptide Enrichment Kit (Tymora Analytical) according to the manufacturer’s instructions.

Techniques: Mutagenesis, Liquid Chromatography with Mass Spectroscopy, Comparison, Saline

Journal: bioRxiv

Article Title: LRRK2 mediates haloperidol-induced changes in indirect pathway striatal projection neurons

doi: 10.1101/2024.06.06.597594

Figure Lengend Snippet: A. Results of GSEA for the Gene Ontology: Biological Processes gene set on genes with at least one significantly differentially regulated phosphopeptide for the WT haloperidol vs vehicle treated comparison. All pathways displayed are significantly differently regulated (adjusted p-value < 0.05 by Fisher’s test). Length of bars shows the number of genes in the pathway whose phosphostate is differentially regulated, and bars are shaded by adjusted p-value. Highlighted pathways are related to known LRRK2 functions. B. Correlation plot for , comparing GS vehicle/WT vehicle to WT haloperidol-vehicle effect size. All points mapped, all significant phosphopeptides (|Log2FC| > 2 and p-value < 0.05 by multiple unpaired t-tests) for either comparison are highlighted and used for correlation. Blue line represents the line of best fit. C. Volcano plot of the striatal proteome comparing the haloperidol-vs. vehicle-treated WT mice. Differentially regulated proteins (|Log2FC| > 2 and p-value < 0.05 by multiple unpaired t-tests) are colored in red and blue for up- and downregulated, respectively. D. Heatmap of effect size (Log2FC) of either haloperidol treatment or LRRK2-GS for all proteins differentially expressed (|Log2FC| > 2 and p-value < 0.05 multiple unpaired t-tests) compared to the vehicle-treated wild type condition in the wild type haloperidol-vs vehicle-treated comparison, mapped for both the wild type haloperidol-vs vehicle-treated comparison and the GS vs. wild type vehicle-treated comparison. Each bar represents a protein E. Volcano plot of relative phosphopeptide for haloperidol- and vehicle-treated LRRK2-GS mutant mice. Phosphopeptides that are differentially regulated (|Log2FC| > 2 and p-value < 0.05 by multiple unpaired t-tests) are colored in red and blue for up and downregulated, respectively.

Article Snippet: The samples were dried completely in a vacuum centrifuge and used for phosphopeptide enrichment using PolyMAC Phosphopeptide Enrichment Kit (Tymora Analytical) according to the manufacturer’s instructions.

Techniques: Comparison, Mutagenesis