ABSTRACT: Bottom-up mass spectrometric identification of interaction partners of candidate microproteins-GFP fusions after anti-GFP pulldown and tryptic digest
Sample preparation
For each of the conditions, we prepared two biological replicates. For each replicate, a confluent T75 flask of cells was pelleted for five minutes at 300 x g. Pellets were washed with PBS (Sigma-Aldrich, D8537), the suspension centrifuged for five minutes at 300 x g again and the resulting pellet flash frozen in dry ice and Methanol (VWR, 20847.307). Pellets were thawed on ice, resuspended in ice-cold RIPA buffer (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.25 % sodium deoxycholate, 1mM EDTA, 1% NP-40) supplemented with Complete Protease inhibitor (Sigma, 5056489001) and incubated for five minutes on ice. The resulting protein extracts were centrifuged at maximum speed on a table-top centrifuge for 15 minutes at 4C and the supernatant (soluble fraction) incubated with 25 ul GFP-trap magnetic beads (ChromoTek, gtma) for four hours at 4C under constant rotation. The protein-bound beads were then washed once with RIPA buffer, twice with PBS or wash buffer (0.5 % NP-40, 0.1 mM EDTA, 20 mM Tris-HCl pH 7.4, 500 mM NaCl) and once with ddH20. Bound proteins were subsequently eluted twice for five minutes with 15 ul 1% acetic acid. Protein eluates were reduced and alkylated in the same step with 5 mM TCEP (Thermo, PG82080) and 20 mM chloroacetamide (Sigma, C0267) in 250 mM Tris buffer pH 8 for 30 minutes at room temperature. Subsequently, Sera-Mag magnetic bead mix (1:1 ratio of Sigma, GE45152105050250 and GE65152105050250) was added in a 25:1 protein to bead volume ratio and proteins were bound onto the beads by adding an equal volume (protein plus beads) of absolute ethanol. After a five minute incubation, the supernatant was removed and beads were washed three times with 80% ethanol. To perform on-bead digestion, protein-bound beads were resuspended in 50mM TEAB pH 8 (Sigma, T7408) containing 1 ug trypsin (Thermo, 90057) and incubated overnight gently shaking at 37C. Afterwards, beads were allowed to settle on the magnetic rack and the supernatant was taken and kept as the first protein eluate. Subsequently, another elution was performed by adding 50 ul 2% acetonitrile (Sigma, 34851) in 50 mM TEAB. The second elution was combined with the first one and the eluate was dried in a SpeedVac.
Simon Elsasser 
