SCL of 4502 find more proteins encoded by the SD1 genome was predicted using the bioinformatic algorithms PSORTb, SignalP, TatP, TMHMM, BOMP, LipoP and KEGG. 350 outer and inner membrane proteins corresponding to ca. 38% of the SD1 membrane proteome, and 1410 cytoplasmic and periplasmic proteins representing ca. 39% of SD1 soluble proteins were identified. Highly abundant SD1 proteins, in vivo and in vitro, were implicated in energy/carbon metabolism and protein synthesis. This included glycolytic enzymes AZD4547 mw (PckA, GapA, Tpi, Fba,
Pgk, GpmA, Eno), elongation factors (FusA, TufA, Tsf), several ribosomal protein subunits (RpsD/K/M, RplC/D/E, RpmC/D/J), and stress response proteins (WrbA, AhpC, SodB). Proteins with global regulatory functions in the cellular stress response were identified in vivo as well as in vitro (Hns, RpoS and CpxR). In summary, SD1 cells produced proteins essential for growth and cell integrity (energy generation, protein synthesis, cell envelope structure) as well as response to cellular and environmental stresses in high abundance. Differential selleck chemicals llc abundance analyses of the SD1 in vitro and in vivo proteomes Data from three biological replicates pertaining to in vivo and in vitro conditions were subjected to statistical analyses. The biological replicate analyses were pooled for the Z-test, and analyzed separately by the SAM test. Differential expression
analysis of the in vitro vs. in vivo proteomes using a two-tailed Z-test resulted in ca. 300 proteins identified as being differentially abundant at a 99% confidence level (Figure 3), while the SAM test identified ca. 90 differentially expressed proteins (Additional File 2, Table S2). As the SAM test takes into account the biological variability between replicates, it is more conservative at estimating the differential protein expression given the dynamic range of the biological data which may inflate variance measures. The Benjamini-Hochberg (B-H) multiple test correction performed on the 1224 proteins common to the in vitro and in vivo samples estimated the FDR at <5% for the ca. 300 differentially expressed
proteins identified from the Z-test (Additional Files 1 and 2, Tables S1 and S2). Hierarchial clustering of the data resulted in several major clusters of similarly expressed MycoClean Mycoplasma Removal Kit proteins (Figure 4). Selection of two clusters magnified in Figure 4 was based on biological interest in the set of proteins that exhibited differential abundance values. For example, one of the clusters harbored numerous ribosomal proteins and several Ipa/Ipg host cell invasion proteins, all of which were clearly increased in abundance in vivo. Another cluster harbored several enzymes indicative of the shift from aerobic to anaerobic energy generation. Protein functional role categories of the differentially expressed proteins were assigned according to the CMR database http://cmr.jcvi.org and are displayed in Figure 5.