Bacterial interactomes: interacting protein partners share similar function and are validated in independent assays more frequently than previously reported
M. Shatsky, S. Allen, B.L. Gold, N.L. Liu, T.R. Juba, S.A. Reveco, D.A. Elias, R. Prathapam, J. He, W. Yang, E.D. Szakal, H. Liu, M.E. Singer, J.T. Geller, B.R. Lam, A. Saini, V.V. Trotter, S.C. Hall, S.J. Fisher, S.E. Brenner, S.R. Chhabra, T.C. Hazen, J.D. Wall, H.E. Witkowska, M.D. Biggin, J.-M. Chandonia, G. Butland
Spectral library for Desulfovibrio vulgaris Hildenborough (DvH) proteins identified in the course of the large-scale mapping of protein-protein interactions using tandem affinity purification (TAP) methods: in addition to DvH peptides, common contaminants and standards that were spiked into washes separating sample injections are also included. DvH wild-type ATCC29579 was genetically engineered to encode locus specific, affinity purification (AP)-tagged fusion proteins using high-throughput construction and electroporation of non-replicating “suicide constructs”, primarily using the Sequence and Ligation Independent Cloning (SLIC) method for plasmid construction. Construct generation and affinity purification methods were previously described (Chhabra et al., 2011. Appl. Environ. Micriobiol. 77:7595-7604). Out of 1401 genes that were successfully fused with AP tag, 957 produced fusion proteins that were detected by MS as baits. In total, 1323 bait and prey proteins were identified by at least two peptides. AP-pulldowns were processed in parallel using gel-free and gel-based workflows. In gel-free workflow protein mixtures were digested with trypsin on PVDF membrane using Millipore 96-well PVDF plates, as described in Chhabra et al., 2011). In gel-based workflow, proteins were fractionated by SDS-PAGE prior to in-gel tryptic digestion. With few exceptions, the majority of gel-free derived samples were analyzed by LC MS using Thermo LTQ Velos Orbitrap. The majority of gel-based samples were analyzed by LC MS using Thermo LTQ XL linear ion trap. Peak lists were extracted from .raw files using the Mascot Distiller 188.8.131.52 software (Matrix Science). Data were searched with an in-house Mascot version 2.2.04 search engine (Matrix Science) against a custom protein database containing all potential protein products generated via 6-frame translation of the D. vulgaris genome supplemented by frequently observed contaminants and concatenated with the decoy database generated by reversing all D. vulgaris protein sequences (102,572 sequences; 9,848,210 residues) (Elias and Gygi, 2007. Nat Methods 4:207-214).