MassIVE MSV000098836

Partial Public PXD067386

Proximity labeling proteomics of Rac1 and palmitoylation-deficient Rac1 C178S in mouse heart

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Description

Proximity labeling proteomics. Proximity labeling proteomics were performed using the BioID2 fusion protein system (49). Mice were injected with AAV9s encoding troponin T promoter-driven BioID2 fusion proteins (myc-BioID2-Venus-CAAX as control, myc-BioID2-Rac1-WT, and myc-BioID2-Rac1-C178S) at postnatal day 6. At 2 months of age, mice were fed ad libitum with high biotin chow (Teklad, Cat #TD.02458). Forty-eight hours post diet change, mice were sacrificed and hearts excised, rinsed in ice-cold PBS, and flash frozen in liquid nitrogen. Left ventricular tissue was homogenized in modified RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, pH 7.5) in a beadmill homogenizer (Next Advance) and sonicated. Lysates were cleared by centrifugation at 12,000 rpm for 10 minutes at 4C. Lysates (4.5 mg) were incubated with streptavidin-coated magnetic beads (Fisher Scientific, Cat #88817) with end-over-end rotation at 4C for 18-20 hours then washed twice in modified RIPA buffer, once in 1 M KCl, 0.1 M Na2CO3, 2% SDS in Tris pH 7.5, and then in 2 M urea in 10 mM Tris pH 8.0 followed by two more washes in modified RIPA buffer. Lastly, beads were washed in ice-cold 1X PBS and flash frozen in liquid nitrogen prior to proteomics analysis. On-bead digestion and subsequent liquid chromatography with tandem mass spectrometry (LC-MS/MS) analyses were performed exactly as described previously at the University of Michigan Proteomics Resource Facility (84). Briefly, samples were reduced, alkylated, trypsin digested, and reconstituted samples were labeled with TMT 16-plex reagents (Thermo Scientific, Cat #A44521) and subjected to LC-MS/MS. Sequenced peptides were mapped to the mouse proteome and abundance normalized to total sequenced peptides. A complete table with detected peptides and proteins is available in the online supplement. [doi:10.25345/C5VQ2SP8M] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Rac1, BioID2, palmitoylation, cardiac, heart, cardiomyocytes ; DatasetType:Proteomics

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Principal Investigators:
(in alphabetical order) 		Matt Brody, University of Michigan, USA
Submitting User: jateuber
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