polymac kit  (Tymora Analytical Operations LLC)

Journal: eLife

Article Title: LRRK2 regulates synaptic function through modulation of actin cytoskeletal dynamics

doi: 10.7554/eLife.95987

Figure Lengend Snippet: ( A ) Network graph showing one of the topological clusters extracted from the LRRK2 protein-protein interaction network. Proteins are represented as nodes while protein-protein interactions are represented as edges. Node size is proportional to the degree centrality: the larger the node, the higher the degree, the more interactions the node has in the network. ( B ) Gene Ontology biological-processes (GO-BPs) enrichment; the analysis was performed using g:Profiler, GO-BPs with term size >500 were discarded as general terms, and the remaining terms were clustered based on semantics using the keywords: Postsynaptic (Actin) Cytoskeleton, Lamellipodium, Synaptic Vesicle, Actin Polymerization/Nucleation, DNA Recombination/Repair, Cell/Neuron Projection. ( C ) Upper network graph as in ( A ) highlighting actin cytoskeletal proteins in blue and the proportion over the total (pie chart) and lower network graph as in ( A ) highlighting synaptic proteins in red and the proportion over the total (pie chart). ( D ) Experimental design of phospho-proteomic analysis comparing WT and G2019S knockin mouse striata. Phosphopeptide abundances were normalized to total protein levels. ( E ) Volcano plot of differentially enriched phosphopeptides, expressed as Log2 fold change (FC) between G2019S-KI versus WT. Highlighted peptides correspond to LRRK2 interactors discovered by MS in . ( F ) GO enrichment analysis performed with g:Profiler (accessed on 01/23/2025; version e94_eg41_p11), filtering for GO terms with size <200 to avoid overly general categories. The top five most significant terms from the Cellular Component (CC) and Biological Process (BP) domains are shown. ( G ) Sunburst plot showing enriched SynGO CC categories and the percentage of SynGO genes among the differentially phosphorylated proteins. ( H ) Venn diagrams showing common and unique hits across the three datasets: GFP-LRRK2 interactomics, synaptic cluster, and phospho-MS.

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: Protein-Protein interactions, Knock-In, 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