Proteome

All protein interaction data sets from MIPS [13 (link)], Gene Ontology [43 (link)] and PreBIND were collected as described previously [6 (link)]. The YPD protein interaction data are from March 2001 and were originally requested from Proteome, Inc. . Other interaction data sets are from BIND . A BIND yeast import utility was developed to integrate data from SGD [12 (link)], RefSeq [44 (link)], Gene Registry , the list of essential genes from the yeast deletion consortium [11 (link)] and GO terms [43 (link)]. This database ensures proper matching of yeast gene names among the multiple data sets that may use different names for the same genes. The yeast proteome used here is defined by SGD and RefSeq and contains 6,334 ORFs including the mitochondrial chromosome. Before performing comparisons, the various interaction data sets were entered into a local instance of BIND as pairwise protein interaction records. The MIPS complex catalogue was downloaded in February 2002.
The protein interaction data sets used here were composed as follows. 'Gavin Spoke' is the spoke model of the raw purifications from Gavin et al [7 (link)]. 'Y2H' is all known large-scale [2 (link)-5 (link),10 (link)] combined with normal yeast two-hybrid results from MIPS. 'HTP Only' is only high-throughput or large-scale data [2 (link)-7 (link),10 (link)] The 'Benchmark' set was constructed from MIPS, YPD and PreBIND as previously described [6 (link)]. 'Pre HTMS' was composed of all yeast sets except the recent large-scale mass spectrometry data sets [6 (link),7 (link)]. 'AllYeast' was the combination of all above data sets. All data sets are non-redundant.

An Escherichia coli K12 strain was grown in standard LB medium, harvested, washed in PBS, and lysed in BugBuster (Novagen Merck Chemicals, Schwalbach, Germany) according to the manufacturer's protocol. HeLa S3 cells were grown in standard RPMI 1640 medium supplemented with glutamine, antibiotics, and 10% FBS. After being washed with PBS, cells were lysed in cold modified RIPA buffer (50 mm Tris-HCl, pH 7.5, 1 mm EDTA, 150 mm NaCl, 1% N-octylglycoside, 0.1% sodium deoxycholate, complete protease inhibitor mixture (Roche)) and incubated for 15 min on ice. Lysates were cleared by centrifugation, and after precipitation with chloroform/methanol, proteins were resuspended in 6 m urea, 2 m thiourea, 10 mm HEPES, pH 8.0. Prior to in-solution digestion, 60-μg protein samples from HeLa S3 lysates were spiked with either 10 μg or 30 μg of E. coli K12 lysates based on protein amount (Bradford assay). Both batches were reduced with dithiothreitol and alkylated with iodoacetamide. Proteins were digested with LysC (Wako Chemicals, GmbH, Neuss, Germany) for 4 h and then trypsin digested overnight (Promega, GmbH, Mannheim, Germany). Digestion was stopped by the addition of 2% trifluroacetic acid. Peptides were separated by isoelectric focusing into 24 fractions on a 3100 OFFGEL Fractionator (Agilent, GmbH, Böblingen, Germany) as described in Ref. 45 (link). Each fraction was purified with C₁₈ StageTips (46 (link)) and analyzed via liquid chromatography combined with electrospray tandem mass spectrometry on an LTQ Orbitrap (Thermo Fisher) with lock mass calibration (47 (link)). All raw files were searched against the human and E. coli complete proteome sequences obtained from UniProt (version from January 2013) and a set of commonly observed contaminants. MS/MS spectra were filtered to contain at most eight peaks per 100 mass unit intervals. The initial MS mass tolerance was 20 ppm, and MS/MS fragment ions could deviate by up to 0.5 Da (48 (link)). For quantification, intensities can be determined alternatively as the full peak volume or as the intensity maximum over the retention time profile, and the latter method was used here as the default. Intensities of different isotopic peaks in an isotope pattern are always summed up for further analysis. MaxQuant offers a choice of the degree of uniqueness required in order for peptides to be included for quantification: “all peptides,” “only unique peptides,” and “unique plus razor peptides” (42 (link)). Here we chose the latter, because it is a good compromise between the two competing interests of using only peptides that undoubtedly belong to a protein and using as many peptide signals as possible. The distribution of peptide ions over fractions and samples is shown in supplemental Fig. S1.