Spot (present in all replicates) detection was carried out using Progenesis SameSpots software (Nonlinear Dynamics) and a master gel image was produced. The reproducibility of spot differences
was confirmed by analyzing three gels for each strain, each obtained using an independent culture. Spots of interest were subjected to tryptic in-gel digestion and identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) using a Voyager DE STR Instrument (Applied Biosystems), as previously described [38]. The α-cyano-4-hydroxycinnamic acid matrix was prepared at 4 g l-1 in 0.1% TFA, 50% acetonitrile. An equal volume (1 μl) of matrix and sample were spotted onto the MALDI-TOF target plate.
Spectra were acquired in the reflector mode with this website the following parameters: 2250 laser intensity, 20 kV accelerating voltage, 62% grid voltage, 135 ns delay. The mass gates used were 700-4000 Da. Internal calibration was performed by using the trypsin peptides at 842.5 and 2211.1 Da. Spots mass accuracy varied between 15-30 ppm. The carbamidomethylation of cysteines, methionine oxidation and one miscleavage were considered during the search. A minimum of four matching peptides and a sequence coverage above 25% were required before considering this a result of the database search. Additional parameters were used to assume a correct identification: theoretical molecular weight and isoelectric point in good agreement with experimental
values. Proteins were identified using MS-Fit software (University of California San Francisco Mass Spectrometry Facility; http://prospector.ucsf.edu and Mascot software Epacadostat ic50 (Matrix Science Inc., Boston, MA; http://www.matrixscience.com). The genome database entries of the chromosome of B. longum NCC2705 (GenBank database accession no. AE014295) were used to assign putative genes encoding the cytosolic proteins of interest from the four B. longum extracts using peptide mass fingerprinting. Based on comparison Chloroambucil against the master gel, we identified spots that were not present in all strains, i.e. pattern differences. The presence or absence of a spot (protein) can reflect whether the gene encoding the protein is present, is expressed or repressed, or may reflect a change in the location of the spot on the gel. Our approach resulted in identification of spots (proteins) corresponding to genes in the NCC2705 genome. Aggregation and cell surface hydrophobicity assays The aggregation assay was performed using bacteria grown at 37°C for 48 hrs in TGYH broth that was harvested and resuspended in TGYH at an OD600 of 0.5. During incubation at 37°C, the OD600 of the suspension was monitored at 30, 60, 120 and 180 min, and aggregation was expressed as [1-(OD600 upper suspension/OD600 total bacterial suspension)] × 100 [36]. To assay cell surface hydrophobicity, bacteria were grown in TGYH as described above, washed twice in 10 ml phosphate buffer (pH 6.