To the Editor: In a recent paper, Braun et al.1 compared the ability of five different methods to detect well-known protein-protein interactions (PPIs). The remarkable outcome of this study was that each of the tested methods detected a different subset of interactions with no single method detecting more than 36% of the tested gold-standard interactions (Fig. 1). Use of all five methods was required to find 62 of 92 interactions (67.4%), whereas individual methods detected only 28.8 out of 92 interactions on average (31.3%). Here we report a similar result when using multiple variants of the yeast two-hybrid (Y2H) system instead of multiple different methods. We have shown that different two-hybrid systems detect markedly different subsets of interactions in the same interactome2,3. Therefore we determined whether the five different methods used by Braun et al.1 could be entirely replaced by variants of the Y2H system. This would have the benefit that only one method needs to be established, and the results of the variants are directly comparable. The set of protein pairs of Braun et al.1 provides a perfect benchmark for such a comparison. We cloned the human positive reference set (PRS) and the random reference set (RRS) from Braun et al.1 (92 protein pairs each) into the following yeast two-hybrid bait-prey vectors: pGBGT7g-pGADCg, pGBGT7g-pGADT7g, pDEST32-pDEST22, pGBKCg-pGADT7g and pGBKCg-pGADCg2,3 (Supplementary Data). In addition to each vector pair, we tested each protein both as activation (prey) and DNA-binding domain fusion (bait), including C-terminal fusions in pGBKCg and pGADCg. This way, we tested each protein pair in ten different configurations (Fig. 1 and Supplementary Table 1).