Mass spectrometry data for pulldown of the Abelson first quarter region from Drosophila lysate
The 'first quarter' (1Q) region of the Drosophila Abelson (Abl) C-terminal domain is indispensible for Abl function, and may participate in protein-protein interactions. Here, we use a GST-1Q recombinant protein to isolate binding partners of 1Q from Drosophila embryo lysate, and identify said proteins by liquid chromatography-tandem mass spectrometry.
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GST pulldown Roughly 4ml of frozen overnight collections of embryos were lysed in 20 mL total volume of lysis buffer (0.25% IGEPAL CA-630, 25 mM Tris pH 7.4, 100 mM NaCl, 5% glycerol, 1 mM MgCl2, 1 mM CaCl2, 10 mM NaF, 2 mM Na3VO4 pH 10, 1 mM ATP, 1x Problock Gold protease inhibitor cocktail (Goldbio)) by homogenization in a 15 mL Dounce homogenizer (10 strokes of each pestle), then sonicated for 1 min total sonication time with 5s per pulse and 10s breaks at amplitude 5. Debris was pelleted by centrifugation for 10 min at 7.2k xG. Lysate was consecutively precleared for 20 min in 2ml, 1ml, and 0.5 mL of Glutathione Sepharose 4B beads (GE Healthcare, Cat#17075601). For pulldown, 75 uL Glutathione Sepharose 4B beads were loaded with 200 ug bait protein (GST, GST-1Q). Beads were incubated with 3 mL of lysate for 1.5 hrs at 4°C, then washed 5 times with 500 uL lysis buffer. Co-purified proteins were eluted in 3 x 33uL 50 mM glutathione in lysis buffer (+ 0.3M Tris pH 7.4), and fractions were combined. All experiments were performed in triplicate, with separate preparations of embryo lysate. Samples were run on SDS-PAGE gels, and regions of interest were excised. Mass spectrometry and analysis The gel pieces were washed with water and 25 mM NH4HCO3, 50% ACN for 15 min each. The liquid was removed and the gel pieces were dehydrated in 100% ACN for 5 min. Once the liquid was removed, the gel pieces were rehydrated in 50 mM NH4HCO3. After 5 min., an equal volume of 100% ACN was added and the gel pieces were left to incubate in the solution for 15 min. more. All liquid was then removed and the gel pieces were dehydrated once again in 100% ACN for 5 min. Once the liquid was removed after the incubation, the gel pieces were speed vac’ed dry for 5 min. The following was then performed: reduction with 5 mM DTT, 50 mM NH4HCO3; alkylation with 15 mM IAA, 50 mM NH4HCO3; and overnight digestion with sequencing-grade trypsin (Promega) in 40 mM NH4HCO3, 0.01% Protease Max (Promega), and 1 mM CaCl2. The peptides were separated by reversed-phase chromatography (Acclaim PepMap100 C18 column, Thermo Scientific), followed by ionization with the Nanospray Flex Ion Source (Thermo Scientific), and introduced into a Q-Exactive versus Fusion mass spectrometer (Thermo Scientific). Abundant species were fragmented with high energy collision-induced dissociation (HCID for QEx) or collision-induced dissociation (CID for Fusion). Data analysis was performed using Proteome Discoverer 1.4 (Thermo) which incorporated the Mascot (Matrix Science) and Sequest algorithms (Thermo Fisher). The Uniprot_Dros_Compl_20160407 database was searched for drosophila protein sequences and a reverse decoy protein database was run simultaneously for false discovery rate (FDR) determination. Secondary analysis was performed using Scaffold 4.5.3 (Proteome Software).