Methods Help

Procedures for cloning, expression, induction, purification and analysis of individual bait proteins are based largely on the methods of Veraksa, et al. (Developmental Dynamics 232:827-834, 2005, and manuscript in preparation).

A cDNA sequence encoding each bait molecule was cloned into the pMK33-C-TAP-FLAG-HA-BD vector (constructed by the Berkeley Drosophila Genome Project) as a full-length open reading frame fused to a carboxyl-terminal FLAG-HA tag cassette under the control of a metallothionein promoter.  All inserts are full-length sequence-validated.  Each purified clone was used to transiently transfect S2R+ cells (Yanagawa, et al., J. Biol. Chem, 273:48, 32353-32359, 1998) in a 54ml culture.

The individual S2R+ cultures were induced to produce FLAG-HA fusion protein by treatment with media containing 0.35 mM Cu₂SO₄ for 20 hours, after which the cells were lysed in 1X Lysis Buffer (50 mM Tris, 5% glycerol, 0.2% IGEPAL, 1.5 mM MgCl₂, 125 mM NaCl, 25 mM NaF, 1 mM Na₃VO₄, pH 7.5) containing protease inhibitor (Roche cat. #11836145001), passed through a 0.45micron Durapore filter (Millipore SLHV033NB), and HA-affinity purification was performed as follows.  Lysates were incubated overnight with monoclonal anti-HA-agarose beads (Sigma cat. #2095, Clone HA-7), and the beads were washed several times with 1X Lysis Buffer followed by PBS, pH 7.4 (GIBCO cat. #10010), and protein complexes were eluted with a 250ug/ml solution of HA peptide (YPYDVPDYA, BioSynthesis, Inc.) in PBS.  Eluted proteins were precipitated with 20% trichloroacetic acid, then processed for LC-MS.  Protein expression was analyzed by immunofluorescence, denaturing gel electrophoresis and western blotting.

Purified sample was subjected to in-solution trypsin digestion overnight. Tryptic peptides were analyzed by liquid chromatography MS/MS (LC-MS/MS). Peptides were separated across a 45-min gradient ranging from 10% to 35% (v/v) acetonitrile in 0.1% (v/v) formic acid in a microcapillary (125 µm X 18 cm) column packed with C₁₈ reverse-phase material (Magic C18AQ, 5 µm particles, 200 Å pore size, Michrom Bioresources) and on-line analyzed on The LTQ XL (ThermoFisher). For each cycle, one full MS scan was followed by ten MS/MS spectra on the linear ion trap XL from the ten most abundant ions. MS/MS spectra were searched using the Sequest algorithm against Drosophila Melanogaster protein database. All peptide matches were filtered based on tryptic state, Xcorr and dCorr.