1054 Nucleic Acids Research, 2005, Vol. 33, No. 3 Figure 2. Main steps of the analysis for a four-organism group. Each step is detailed in Materials and Methods. After performing the analysis on each four-organism group, the number of groups that resulted in significant P-values was compared with that expected at random in order to obtain the significance of the overall analysis. Significant correlation between predicted protein abundance profiles of related proteins For many four-organism groups, the functionally related pro- teins had statistically signiﬁcantly higher correlation coefﬁ- cients than expected at random (Table 1). The statistical signiﬁcance was evaluated by comparing these correlation coefﬁcients with those obtained in the negative set, using a signiﬁcance threshold of 0.05 (see Figure 2 and Materials and Methods). We looked at groups of organisms for which a negative set of at least 1000 pairs of proteins could be created. This resulted in 15 978 groups of four organisms for the protein–protein interaction data and 69 433 groups of four organisms for the pathway data. For the interacting pairs, 58% of the four-organism groups that were analyzed had stat- istically signiﬁcantly higher correlation coefﬁcients than expected at random (i.e. 58% of the groups differed from random at a signiﬁcance cutoff of 0.05). For the pathway pairs, 42% of the four-organism groups had statistically sig- niﬁcantly higher correlation coefﬁcients than expected at ran- dom (Table 1). These fractions are signiﬁcantly higher than the fraction of 5% that can be expected at random (P 1 · 10 10, using the binomial test). This demonstrates that for many combinations of organisms, the predicted protein abundance proﬁles of related proteins are indeed correlated. When looking at subgroups of organisms (Table 1), the high fraction of subgroups from g -proteobacteria that have Table 1. Correlation between predicted abundance profiles of functionally related proteins for different organism groups Number of organisms in analysis Number of organism groupsa Number (%) of organism groups with P < 0.05 P-valueb 1 · 10 10 1 · 10 10 1 · 10 10 Nrd 1 · 10 10 1 · 10 10 1 · 10 10 0.02 Protein–protein interactions All (48) g-Proteobacteria (7) Gram-positivec (11) Archaea (8) Pathways All (48) g-Proteobacteria (7) Gram-positivec (11) Archaea (8) 15 978 35 70 0 69 433 35 70 70 9253 (58%) 34 (97%) 45 (64%) 0 29 277 (42%) 30 (86%) 41 (59%) 8 (11%) aGroups of four organisms, for which a negative set of at least 1000 protein pairs could be generated, were included in the analyses (see Materials and Methods). bP-values, estimating whether the number of organism groups with P < 0.05 is significant, were calculated using a binomial test, comparing the observed number of groups having P-value < 0.05 with the expected fraction (0.05). cLow G+C Gram-positive bacteria. dNot relevant, since there were no groups with a large enough negative set for statistical evaluation. correlated proﬁles is striking, while for archaea, which are more distantly related to the reference organism E.coli, the phenomenon is quite weak. The ﬁnding that the majority of groups of organisms from g -proteobacteria showed that the