Figure 3. Detection of p50, RelA, RelB, IkBa and GAPDH, and any pairwise interactions in cell lysates. a) Cells were transfected with genes encoding proteins of the NFkB family; either p50, IkBa and RelB (sample I); or p50, IkBa and RelA (sample II). The two populations of cells were lysed and the PLA probes were added to the cell lysates in separate reactions. After generating the reporter molecules by ligation, the two samples were pooled to amplify the ligation products by PCR, followed by readout via DTM. b) The results are represented in a heat map where negative log2 Cy5/Cy3 ratios (color coded as more green) indicate a higher abundance of proteins or protein interactions in sample I than in sample II, while positive log2 Cy5/Cy3 ratios (color coded as more red) indicate the opposite situation. doi:10.1371/journal.pone.0040405.g003 Profiling Protein Complexes by PLA with DTM Microarray analysis of ligation products was performed after first releasing ligated circular DNA from transfected and untransfected cells through the addition of proteinase K. The ratio of the two fluorophores thus reflects the relative SMAD4 abundance in the two cell populations. b-actin protein was detected in the same reactions for normalization. Figure 4c shows the SMAD4 expression ratios for transfected versus untransfected cells, including dye-switch controls. As expected, PLA with DTM readout revealed that transfected cells expressed greater amounts of SMAD4 compared to untransfected cells. Discussion We have previously demonstrated that PLA offers highly sensitive and specific detection of proteins and their interactions over wide dynamic ranges either in solution or in situ, and the method can be applied either for transfected or normally expressed target proteins. Interactions are dynamic processes that are preferentially detected in their natural environment. In this paper we have established a scalable method using microarrays for recording levels of proteins and all pairwise interactions among a targeted set of proteins. The method has been applied for investigating four proteins of the NFkB family along with one housekeeping protein. We demonstrate that the approach can be adapted to different sample types by analyzing both cell lysates and fixed cell samples, and the assays should thus be suitable for analysis of clinical samples such as plasma, serum or other bodily fluids, as well as fixed cells or tissue sections. By using DNA barcodes to represent the targeted proteins, and taking advantage of efficient techniques for nucleic acid analysis, the method allows analysis of the entire interaction space among the set of targeted proteins. This opens up unique possibilities for studying all possible interactions and posttranslational modifications among large groups of proteins, either for research or diagnostic purposes. Also larger complexes of interacting proteins can be investigated by identifying pairwise interactions. Both quantitative real time PCR and next generation sequenc- ing have previously been used to read out multiplex PLA experiments [17–20]. Recently, Lundberg et al [19] developed four 24-plex PLA reactions, using a microfluidic system from Fluidigm to conveniently run real time PCR in chips with 96 amplification reactions for each of 96 DNA samples per chip. Since the potential for binary interaction analysis scales as the square of the number of investigated targets, DTM offers an attractive alternative for inexpensive high-throughput readout. By using two-color DTM analyses, distinctly bar-coded PLA ligation products from pairs of samples can be pooled prior to PCR amplification, allowing the ratios for the corresponding analytes in the two samples to be determined even when array features are saturated, thus achieving a broad dynamic range. The proposed method has limitations for absolute quantification of the abun- dance of proteins or protein-protein interactions, but the method is excellently suited to screen for quantitative differences between pairs of samples. It could be of great value to use the method to screen for differences in levels of proteins and their binary interactions between e.g. cancer and normal tissue, and to follow this by qualitative in situ PLA analyses of the individual or interacting proteins using the same reagents. A main rate limiting factor for further multiplexing of the assay will be the identification of high quality affinity reagents. The quality of the antibodies strongly influences both the sensitivity and the specificity of the assays. We have applied a strict validation pipeline for all binders used in the assays shown herein, to ensure that the antibodies are specific and PLoS ONE | www.plosone.org 5 July 2012 | Volume 7 | Issue 7 | e40405