polymac enrichment kit  (Tymora Analytical Operations LLC)

Journal: bioRxiv

Article Title: Leucine-rich repeat kinase 2 impairs the release sites of Parkinson’s disease vulnerable dopamine axons

doi: 10.1101/2025.08.28.672006

Figure Lengend Snippet: A . WB analysis of SNc extracts from mice previously treated with MLi-2 (10 mg/kg) or vehicle for 2 h and probed for pS106 RAB12 (LRRK2 kinase target), total RAB12, and β-actin. B. Quantification of p-RAB12 band intensities normalized to total RAB12. n=5 mice/condition. Data are represented as mean±SEM (error bars). Asterisks denote statistical significance for Šídák’s multiple comparison tests after two-way ANOVA. *p< 0.05. (Treatment factor F(1,12)=8.614 p=0.0125, Genotype factor F(1, 12)=10.64, p=0.0068) C. Gene Ontology analysis of proteins with at least one differentially regulated phosphopeptide in striatal synaptosomes from vehicle vs MLi2-treated LRRK2 G2019S mice. The top 15 pathways significantly enriched in the cellular component term analysis (adjusted p -value ≤ 0.05 by hypergeometric test with HB correction). All enriched pathways have been uploaded to Zenodo. Link in the resources table. D. Volcano plot comparing the significantly altered total proteins (|Log2FC| > 0.58 and unadjusted p -value ≤ 0.05 by multiple unpaired t-tests) between vehicle and MLi-2-treated LRRK2 G2019S striatal synaptosomes. E . Heat map of the expression of RAB3 isoforms in a dataset of single cell RNA sequencing. The differential expression of RAB3 isoforms in TH+ SNc neurons compared to the remaining non-TH SNc neurons is expressed as normalized mean log values (RAB3A = 4.11, RAB3B = 0.693, RAB3C = 5.59, and RAB3D = 0.693). F. Heat map illustrating the expression of LRRK2 and RAB3B isoform in human dopamine neuron subpopulations from a single nucleus RNA sequencing dataset, plotted with Shinny Cell. 931 G. Workflow of APEX2 experiment for dopamine neuron subpopulation-specific analysis with subcellular compartment resolution. Cre-dependent APEX2 expressing AAV (AAV5-CAG-DIO-APEX2-NES) was injected into the SNc of Anxa1 Cre LRRK2 WT mice for Anxa1+ dopamine neuron-specific APEX2 labeling. H. Relative expression of RAB3 protein isoforms in our Anxa1+ dopamine axon subcluster proteomic dataset (for further details see ) . I. Equal amounts of eluted proteins from streptavidin pulldowns from the striatum and SNc from either a LRRK2 WT or a LRRK2 G2019S DAT Cre mouse injected with APEX2 AAV . J. HEK293T cells were transiently transfected with either pCMV or HA-tagged RAB3 isoforms and blotted with isoform-specific antibodies or anti-HA, and with GAPDH for loading control. Equal expression of all isoforms is shown in the panel below.

Article Snippet: The samples were dried completely in a vacuum centrifuge and subjected to phosphopeptide enrichment using PolyMAC Phosphopeptide Enrichment kit (Tymora Analytical) according to manufacturer’s instructions, and the eluted phosphopeptides dried completely in a vacuum centrifuge.

Techniques: Comparison, Phospho-proteomics, Expressing, RNA Sequencing, Quantitative Proteomics, Injection, Labeling, Transfection, Control

Journal: bioRxiv

Article Title: Leucine-rich repeat kinase 2 impairs the release sites of Parkinson’s disease vulnerable dopamine axons

doi: 10.1101/2025.08.28.672006

Figure Lengend Snippet: A. Experimental workflow of LC/MS-MS analysis in striatal synaptosomes isolated by subcellular fractionation from 3 technical replicates, with each technical replicate performed using 3 LRRK2 G2019S mice treated with either MLi-2 or the corresponding vehicle for 2 hours. Treatment conditions: Vehicle for MLi-2, 40% 2-hydroxypropyl-β-cyclodextrin; MLi-2, 10 mg/kg. This treatment leads to a decrease in LRRK2 kinase activity in vivo (see ). B. Volcano plot comparing the significantly altered phosphopeptides between vehicle and MLi-2-treated LRRK2 G2019S mice (p≤ 0.05 by multiple unpaired t-tests, |Log 2 FC|> 0.58). C . Gene Ontology analysis of proteins with at least one differentially regulated phosphopeptide in striatal synaptosomes from vehicle vs MLi2-treated LRRK2 G2019S mice. The top 15 pathways are significantly enriched in the biological process term analysis. All enriched pathways have been uploaded to Zenodo (link in the key resources table) and presented in Supplementary File 1. D. Representative PhosTag and SDS PAGE gels (upper and lower panels, respectively) from two independent experiments of LRRK2 WT and LRRK2 G2019S mice (indicated WT, GS, respectively) with or without MLi-2 treatment (10 mg/kg, 2 hours). SNc extracts from these mice were probed for RAB3A (closed/open circles = phosphorylated (p)- RAB3A/unphosphorylated RAB3A species, respectively). E. p-RAB3A/total (phosphorylated +unphosphorylated) RAB3A quantification. n=10 mice/treatment. All samples were normalized to the average signal of all WT samples in the same blot. F. Same as D, but probing for RAB3C. G. Same as E, but for RAB3C. n=7 mice/treatment. In E and G, data represent mean±SEM. Asterisks indicate statistical significance, as determined by Šídák’s multiple comparisons test following a two-way ANOVA. E: Treatment factor F(1,27)=11.33 p=0.0023, Genotype factor F(1,27)=8.593 p=0.0068. G, Treatment factor F(1,18)=7.398 p=0.0140, Genotype factor F(1,18)=14.76 p=0.0012. *p<0.05, **p<0.01.

Article Snippet: The samples were dried completely in a vacuum centrifuge and subjected to phosphopeptide enrichment using PolyMAC Phosphopeptide Enrichment kit (Tymora Analytical) according to manufacturer’s instructions, and the eluted phosphopeptides dried completely in a vacuum centrifuge.

Techniques: Liquid Chromatography with Mass Spectroscopy, Isolation, Fractionation, Activity Assay, In Vivo, Phospho-proteomics, SDS Page

Journal: bioRxiv

Article Title: Leucine-rich repeat kinase 2 impairs the release sites of Parkinson’s disease vulnerable dopamine axons

doi: 10.1101/2025.08.28.672006

Figure Lengend Snippet: A. Peptide fragment ions of the phosphopeptide; YR T ITTAYR, of the 2+ precursor ion (B). which spans phosphorylation site T86 in recombinant p-Flag-RAB3A protein. All chromatograms elute at the same retention time, with peptide identification at a false-discovery rate of <1% (DIA-NN 1.9.2).

Article Snippet: The samples were dried completely in a vacuum centrifuge and subjected to phosphopeptide enrichment using PolyMAC Phosphopeptide Enrichment kit (Tymora Analytical) according to manufacturer’s instructions, and the eluted phosphopeptides dried completely in a vacuum centrifuge.

Techniques: Phospho-proteomics, Recombinant

Journal: bioRxiv

Article Title: Leucine-rich repeat kinase 2 impairs the release sites of Parkinson’s disease vulnerable dopamine axons

doi: 10.1101/2025.08.28.672006

Figure Lengend Snippet: A. Schematic illustrating the localization of Anxa1 dopamine neuron subtype in the mouse SNc and their projection patterns in the striatum. B. Experiment groups of APEX2-based proximity labeling within genetically labeled dopamine neurons in the mouse brain. C . 98% of the top 55 mDA neuron marker genes (e.g., TH and DAT (SLC6A3)) from a publicly available APEX2 proteome dataset (dataset identifier PXD026229 ProteomeXchange Consortium) were detected in all our mass spectrometry samples from DAT Cre ; APEX2 EGFP mice. D. Differential expression comparison of SNc and Striatum APEX+ streptavidin pulldown samples. Proteins colored orange or blue indicated significantly enriched in Striatum vs. SNc, respectively. (|Log2FC| > 0.58 and unadjusted p-value ≤ 0.05 by multiple unpaired t-tests) E. Gene Ontology analysis of proteins with at least one differentially regulated phosphopeptide in striatum from vehicle vs. MLi2-treated Anxa1iCre LRRK2 G2019S mice. BP, Biological Process; CC, Cellular Component refer to categories in GO. All enriched pathways have been uploaded to Zenodo; the link can be found in the key resources table.

Article Snippet: The samples were dried completely in a vacuum centrifuge and subjected to phosphopeptide enrichment using PolyMAC Phosphopeptide Enrichment kit (Tymora Analytical) according to manufacturer’s instructions, and the eluted phosphopeptides dried completely in a vacuum centrifuge.

Techniques: Labeling, Marker, Mass Spectrometry, Quantitative Proteomics, Comparison, Phospho-proteomics

Journal: Science Advances

Article Title: Phosphorylation at serine-260 of Toc33 is essential for chloroplast biogenesis

doi: 10.1126/sciadv.adu4054

Figure Lengend Snippet: ( A ) Purified protein for in vitro kinase assay. Flag-Toc33 lacking the transmembrane domain (ΔTM), Flag-CTR1 kinase domain (KD), and the inactive Flag-CTR1 kinase domain mutant (Flag-CTR1-KD D694E ) were purified from E. coli . ( B ) In vitro kinase assay using 32 P[γATP] radiolabeling. ( C to E ) GFP-Toc33 phosphorylation and abundance correspond to increasing Myc-CTR1 coexpression in protoplasts from (C) WT, (D) etr1-1 , and (E) ein2-5 . Protein samples were separated by SDS-PAGE with and without Phos-tag. ( F ) A phosphopeptide spectrum from tandem mass spectrometry (MS/MS) identifying Ser260 as a Toc33 phosphorylation site by CTR1. The mass shift between y5 and y6 (69 Da) confirms S260 phosphorylation (Ser mass 87 Da + phosphate 80 Da − neutral loss H 3 PO 4 98 Da = 69 Da). ( G ) Phosphorylation of Myc-Toc33-WT, Myc-Toc33-S258A, or Myc-Toc33-S260A mutant proteins by GFP-CTR1 in Arabidopsis protoplasts, analyzed via SDS-PAGE with and without Phos-tag.

Article Snippet: The samples were desalted using a Pierce C18 spin column (Thermo Fisher Scientific) and phosphoenriched using the Spin-Tip PolyMAC-Ti Phosphopeptide Enrichment Kit (Tymora) following the manufacturer’s protocol.

Techniques: Purification, In Vitro, Kinase Assay, Mutagenesis, Radioactivity, SDS Page, Mass Spectrometry, Tandem Mass Spectroscopy