Proteomics of model bacterial communities Harvesting and pelleting of bacteria, proteomic analysis, mass spectrometry and statistical methods were handled as described in Kuboniwa et al.[11]. In brief, bacteria were cultured to mid-log phase, harvested by centrifugation and resuspended in pre-reduced PBS (rPBS). 1 x 109 cells of S. gordonii were mixed with

an equal number AZD0156 ic50 of P. gingivalis, F. nucleatum, or both as combinations of the species. S. gordonii cells alone were also used as a control. Two independent biological replicates from separate experiments comprised of at least two technical replicates were analyzed. Bacteria were centrifuged at 3000 g for 5 min, and pelleted mixtures of bacteria were held in 1 ml pre-reduced PBS in an anaerobic chamber at 37°C for 18 h [10]. Bacterial cells were lysed in resuspension buffer (15 mM Tris HCl pH 9.5, 0.02% Rapigesttm Waters, Milford, MA) in a boiling water bath followed by sonication and bead beating and proteins were digested with trypsin then fractionated into five pre-fractions [33]. The 2D capillary HPLC/MS/MS analyses were conducted on a Thermo LTQ mass spectrometer (Thermo Fisher Corp. San Jose, CA, USA). Peptides were eluted with a seven step salt gradient (0, 10, 25, 50, 100, 250 and 500 mM ammonium acetate) followed by an acetonitrile gradient elution (Solvent A: 99.5% water, 0.5% acetic acid. Solvent B: 99.5% acetonitrile, 0.5% acetic acid). The MS1 scan range

for all samples was 400–2000 m/z. Each MS1 scan was followed by 10 MS2 scans in a data dependent manner for the 10 most intense ions in the MS1 scan. Default parameters under Xcalibur 1.4 high throughput screening data acquisition software (Thermo Fisher) were used, with the exception

of an isolation width of 3.0 m/z units and normalized collision energy of Sucrase 40%. Data processing and protein identification Data processing was handled as described in Kuboniwa et al.[11]. In brief, raw data were searched by SEQUEST [34] against a FASTA protein ORF database consisting of the P. gingivalis W83 (2006, TIGR-CMR [35]) [GenBank: AE015924], S. gordonii Challis NCTC7868 (2007, TIGR-CMR [36]) [GenBank: CP00725.1], F. nucleatum ATCC 25586 (2002, TIGR-CMR [37]) [GenBank: AE009951.1], bovine (2005, UC Santa Cruz), nrdb human subset (NCBI, as provided with Thermo Bioworks ver. 3.3) and the MGC (Mammalian Gene collection, 2004 curation, NIH-NCI [38]) concatenated with the reversed sequences. The reversed sequences were used for purposes of calculating a qualitative FDR using the published method [39, 40]. The SEQUEST peptide level search results were filtered and grouped by protein using DTASelect [41], then input into a FileMaker script developed in-house [42, 43] for further processing, including peak list generation. Only peptides that were unique to a given ORF were used in the calculations, ignoring tryptic fragments that were common to more than one ORF or more than one organism, or both.