Proteomic analysis of a lactic bacterium: level of replication and ANOVA

A proteomic experiment on Lactococcus lactis with both biological and technical replications was carried out. The data of each type of replication were analysed separately by analysis of variance. The results show that many false positives may be detected in the absence of biological replication and suggest that analysis of variance is a useful method to analyse proteomic data. Levels of replication in 2-DE based proteomic experiments may be grouped in two types: (a) biological replication is the replication of biological samples, (b) technical replication is the replication of gels for a given biological sample. Several papers recommend to use biological replications in differential expression experiments (see e.g. [1, 2]). This recommendation is consistent with the results of several studies, which found that biological variance was larger than technical variance in proteomic experiments on plants, animals, E. coli (see e.g. [3, 4, 5]). The first objective of this paper is to quantify the effect of level of replication on the results of proteomic experiments on the bacterium Lactococcus lactis. Analysis of variance (ANOVA) has been proposed to analyse the data of differential expression experiments and has been implemented in several ways, such as gene-by-gene ANOVA or global ANOVA (see e.g. [6, 7, 8, 9, 10, 11, 12]). In a standard ANOVA, observations are assumed to follow independent normal distributions with the same variance. These assumptions may not be fully satisfied in proteomic experiments, even if a transformation of the data has been used [10]. The second objective of this paper is to study if ANOVA is a reliable method to analyse the data of proteomic experiments on L. lactis. The rest of this paper presents the experiment we carried out and the analyses of the data. To compare biological and technical replications, an experiment with both types of replication was carried out. Four precultures of the same strain of L. lactis were used to inoculate four cultures. These cultures were carried out in four flasks, with the same medium, on the same day. One protein extract was prepared from each culture. Then, six 2-DE gels were made: Gels 1 and 2 were made from the protein extract of Culture 1, Gels 3 and 4 from the protein extract of Culture 2, Gel 5 from the protein extract of Culture 3, and Gel 6 from the protein extract of Culture 4 (Fig. 1). The experiment was performed by the same experimenter with a standard protocole. Precultures were considered as biological replications and gels made from the same protein extract were considered as technical replications. The spot volumes of 254 proteins were measured on each gel, and ranged from 106 to 237600 with a median value of 5414. 14% of volumes was missing in the initial data file, presumably because these volumes were too small to be detected. Missing observations were replaced by a small value, namely 100, which was the order of magnitude of the smallest observable volume. This experiment is similar to the uniformity trials which have been used in agriculture and in other contexts [13]. In such experiments, dummy treatments are superimposed to data obtained under uniform conditions, in order to compare design and analysis methods. The false positive rate is the expected proportion of false positives among proteins