2Relative abundance based on normalized total spectral counts. 3Proteins not identified in Experiment II (see Table 4). (ii) iTRAQ To more closely examine and quantify O157 protein expression in the bovine rumen, especially in the uRF, the anaerobic O157-proteome expressed in LB, dRF, fRF and uRF after 48 h incubation was compared using iTRAQ, in Experiment II. Data generated in two runs for each biological replicate was condensed

to create a single comprehensive file per AUY-922 chemical structure sample, and the files for the two biological replicate samples compared (Additional file 2: Table S2) to identify unambiguous proteins. Using the anaerobic O157-proteome expressed in LB as the reference, a total of 394 O157 proteins that were either differentially or similarly expressed in dRF, fRF, and uRF were identified (Figure 3, Additional file 2: Table S2). Of the cumulative 35 O157 proteins expressed anaerobically in dRF and fRF, and identified via Bottom-up proteomics,

10 were not identified using iTRAQ in the second experiment (Table 3). Overall, only 134 Tideglusib mw proteins were common to the results of the two experiments, indicative of incubation-time related differences in the number and type of proteins expressed. Differentially expressed O157 proteins in the iTRAQ dataset distributed as 298/394 in dRF (169, up-regulated, 129, down-regulated), 241/394 in fRF (162, up-regulated, 79, down-regulated) and 237/394 in uRF (155, up-regulated, 82, down-regulated) (Table 4). Interestingly,

PIK3C2G similar expression patterns were observed between O157 proteins expressed in dRF and uRF; 90% of dRF-differentially regulated and 71% dRF-no change proteins were similarly expressed in uRF. This may have been due to shared growth conditions (nutrient limitation)/signals in these two media. The competing microflora in uRF may have decreased nutrients in that media. Figure 3 Log fold changes in the expression of O157 proteins, identified using iTRAQ, in media tested under anaerobic conditions. The O157-proteome expressed in LB was the reference against which the regulation of O157 proteins in other media was determined. The scatter plots represent O157 proteins expressed in the context of the 155 up-regulated in uRF (Panel A), 82 down-regulated in uRF (Panel B) and 157 with no change in expression levels in uRF (Panel C). LB, Luria-Bertani broth; dRF, depleted and filtered rumen fluid; fRF, filtered rumen fluid; uRF, unfiltered rumen fluid.