Project PXD001877

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Arabidopsis endomembrane enrichments: Affinity enrichment of YFP-GOT1


Our aim was to dissect subcellular trafficking routes by enriching for partially overlapping subpopulations of endosomal proteomes associated with endomembrane markers. We selected RABD2a/ARA5, RABF2b/ARA7, RABF1/ARA6, and RABG3f as markers for combinations of the Golgi, trans-Golgi Network (TGN), Early Endosomes (EE), secretory vesicles, Late Endosomes (LE), Multivesicular Bodies (MVB) compartments and tonoplast. As comparisons we used Golgi transport 1 (GOT1), which localises to the Golgi, Clathrin Light Chain 2 (CLC2) labelling Clathrin-coated vesicles and pits and the Vesicle Associated Membrane Protein 711 (VAMP711) present at the tonoplast. We developed an easy-to-use method by refining published protocols based on affinity purification of fluorescent fusion constructs to these seven subcellular marker proteins in Arabidopsis thaliana seedlings.

Sample Processing Protocol

Protein Extraction and Affinity Purification – For all constructs, eight day old seedlings of A. thaliana seedlings were harvested and frozen in liquid nitrogen and ground with a pestle and mortar. Protein extraction buffer (150 mM Na-HEPES pH7.5, 10 mM EDTA, 10 mM EGTA, 17.5% (w/v) sucrose, 7.5 mM KCl, 0.01% (v/v) Igepal, 10 mM DTT (Dithiothreitol), 1% (v/v) Protease inhibitors (Sigma), 0.5% (v/v) polyvinylpolypyrrolidone) at 2 ml to 1 g of fresh weight tissue was added. All subsequent steps were performed at 4 oC. Protein concentration was determined with BCA assay and BSA as the standard. Homogenate was filtered through two layers of miracloth and centrifuged at 6000 g for 20 min. 20 μl of chromotek GFP or RFP trap sepharose beads (as appropriate) were added per 50 ml homogenate and incubated for 3 hours with shaking. The homogenate was then centrifuged at 500 g for 5 min and the supernatant discarded. The bead slurry was washed 5 times with fresh pre-chilled extraction buffer (no polyvinylpolypyrrolidone or protease inhibitors) with 3 min incubation. The slurry was collected after the last wash and protein eluted with 2x SDS-PAGE loading buffer and taken for either LC-MS or Western blotting. Tryptic digestion of proteins – Affinity purified proteins were separated on 4-20% Tris-Glycine nUView pre-cast gradient gels (NuSep). The SDS-PAGE gels were cut into 7 slices per affinity purification. Gel slices were washed for 30 mins with 50% ACN/ 25 mM ammonium bicarbonate (ABC) at 37 oC, twice. Then 100% ACN was added for 10 mins and the liquid removed. 10 mM DTT in 50 mM ABC was added to cover the gel pieces for 30 min at 56oC shaking and the supernatant removed. 55 mM Chloroacetamide in ABC (in the dark) was applied for 20 mins. The gel pieces were washed twice for 15 min with 50% ACN/ 25 mM ABC and dehydrated with 100% ACN for 10 mins. 1μg of trypsin, 46 mM ABC, 5% ACN was applied at 37 0C overnight and the supernatant removed and retained. The gel pieces were washed three times by addition of 50% ACN, 5% formic acid and sonicated for 10 mins and the wash supernatants were then pooled with previous supernatants. The supernatants containing the peptides were then dehydrated to dryness. The control samples, from plant expressing the fluorescent tags mCherry or GFP only were treated as above expect that the tryptic digestions were performed from the affinity beads directly, and these samples were analysed on an Orbitrap Fusion, not an Orbitrap XL.

Data Processing Protocol

Software processing and peptide identification - Peak lists in format of Mascot generic files (.mgf files) were prepared from raw data using Proteome Discoverer v1.2 (ThermoFisher Scientific). Peak picking settings were as follows: m/z range set to 300-5 000, minimum number of peaks in a spectrum was set to 1, S/N threshold for Orbitrap spectra set to 1.5, and automatic treatment of unrecognized charge states was used. Peak lists were searched on Mascot server v.2.4.1 (Matrix Science) against TAIR (version 10) database with GFP, RFP and common contaminants such as keratin added. Only tryptic peptides, were permitted with up to 2 possible miscleavages and charge states +2, +3, +4, were allowed in the search. The following modifications were included in the search: oxidized methionine (variable), carbamidomethylated cysteine (static). Data were searched with a monoisotopic precursor and fragment ions mass tolerance 10 ppm and 0.8 Da respectively. Mascot results were combined in Scaffold v. 4 (Proteome Software) and exported in Excel (Microsoft Office). Peptide identifications were accepted if they could be established at greater than 95.0% probability by the Peptide Prophet algorithm with a minimum of two unique peptides at 99% PeptideProphet probability {Searle, 2010 #111}.


Alex Jones, University of Warwick
Alexandra M. E. Jones, School of Life Sciences, University of Warwick ( lab head )

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    Heard W, Sklenar J, Tome DF, Robatzek S, Jones AM. Identification of regulatory and cargo proteins of endosomal and secretory pathways in Arabidopsis thaliana by proteomic dissection. Mol Cell Proteomics. 2015 Apr 21. pii: mcp.M115.050286 PubMed: 25900983


Showing 1 - 3 of 3 results
# Accession Title Proteins Peptides Unique Peptides Spectra Identified Spectra View in Reactome
1 46750 no assay title provided (mzIdentML) 8007 22623 9550 13614 13614
2 46751 no assay title provided (mzIdentML) 5358 16083 6812 9430 9430
3 46752 no assay title provided (mzIdentML) 13552 27869 15603 19443 19443