For each Rab column, 5E8 S2 cells were harvested by centrifugation for lysis.
Each of the 23 D. melanogaster Rabs that have a mammalian ortholog were expressed as recombinant GST fusions (in E. coli) bearing the Q>L mutation that is known to stabilize the GTP-bound form (constitutively active). GST-Rab fusions were coupled to glutathione sepharose and incubated with clarified S2 cell lysates (10-20mg/ml protein extract) that were prepared using CHAPS detergent, in the presence of non-hydrolysable GTP analogue GppNHp. Columns were washed and bound proteins were eluted using high salt and GDP. Proteins were concentrated by chloroform/methanol protein precipitation, separated by SDS-PAGE, and proteins >45 KDa were excised for subsequent identification.
Purified proteins were identified by liquid chromatography-mass spectrometry. Peptide searches were against FlyBase Dmel 5.9.
Peptide searches were against FlyBase Dmel 5.9, and protein identifications were accepted if they could be established at >95.0% probablility and contained at least two identified peptides. The predicted false discovery rate (FDR) was less than 1%. Spectral counts were used to estimate protein abundance. An "S-score" was calculated for each interaction, which gives greater weight to interactions seen with fewer baits.
The use of detergent during S2 cell lysis solubilized more proteins, which allowed for the recovery of more known Rab interacting proteins, but also increased the number of non-specifing binding proteins isolated across the parallel affinity purifications. Using detergent-free cell lysis in the related Rab_Effectors_2 dataset resulted in a cleaner dataset, but some known Rab-binding proteins were lost or reduced.
