Biological versus technical variability in 2‐D DIGE experiments with environmental bacteria

Abstract

Two-dimensional difference gel electrophoresis (2-D DIGE) allows for reliable quantification of global protein abundance changes. The threshold of significance for protein abundance changes depends on the experimental variation (biological and technical). This study estimates biological, technical and total variation inherent to 2-D DIGE analysis of environmental bacteria, using the model organisms "Aromatoleum aromaticum" EbN1 and Phaeobacter gallaeciensis DSM 17395. Of both bacteria the soluble proteomes were analyzed from replicate cultures. For strains EbN1 and DSM 17395, respectively, CV revealed a total variation of below 19 and 15%, an average technical variation of 12 and 7%, and an average biological variation of 18 and 17%. Multivariate analysis of variance confirmed domination of biological over technical variance to be significant in most cases. To visualize variances, the complex protein data have been plotted with a multidimensional scaling technique. Furthermore, comparison of different treatment groups (different substrate conditions) demonstrated that variability within groups is significantly smaller than differences caused by treatment.

Gene Regulatory and Metabolic Adaptation Processes of Dinoroseobacter shibae DFL12T during Oxygen Depletion

“…Pairwise comparisons of obtained expression rates for different time points after the shift to anoxic conditions with data obtained prior to anaerobic shift (0 min) were made ( Table 1). The investigated time points were 5,10,15,20,30,60, and 120 min after the oxygen supply was switched off (Fig. 1).…”

“…Isoelectric focusing (pH range of 3.0 -5.6 and 3-11 non-linear) and second dimension separation were performed as described previously (13). Spots fulfilling the following criteria were considered to have significantly different abundances: -fold change of ՅϪ1.5 or Ն1.5 (20), an analysis of variance p value of Ͻ0.05, t test value of Ͻ10 Ϫ4 , and matched in at least 75% of the analyzed gel images.…”

Carbohydrate Catabolism in Phaeobacter inhibens DSM 17395, a Member of the Marine Roseobacter Clade

“…The detection parameters were set as described previously (30). Those protein spots that changed in abundance by ՆԽ1.5Խ-fold, matched in at least 75% of the gels analyzed, and had P values of Ͻ0.05 by analysis of variance (ANOVA) and t test values of Ͻ10 Ϫ4 were regarded as significantly regulated (31). For protein identification, separate replicate 2DE gels with 300-g protein loads were run for each substrate condition and were stained with colloidal Coomassie brilliant blue (cCBB) after electrophoresis (30).…”

Proteomic Analysis of the Cerebrospinal Fluid of Parkinson's Disease Patients Pre- and Post-Deep Brain Stimulation

“…The CVi were calculated from the SA(standardized abundance)-values of protein spots across different sets of gels. The median of CVi values per used gel set yields the biological variation [20]. …”