Figure 1. Schematic illustration of PLA analysis with DTM readout. a) The PLA probes, each carrying unique DNA sequences (A1, B1, …, N1, or A2, B2, …, N2) that code for their target antigens, are incubated with the samples to be examined. b) The oligonucleotides on pairs of PLA probes that have bound their targets in close proximity are ligated to give rise to reporter DNA strands. In the ligation step a sample barcode is introduced in the ligation product to allow for dual color comparisons of results for two samples in the same array spot. c) The ligation products are amplified by PCR and treated with DNA modifying enzymes to generate single stranded reporter molecules Profiling Protein Complexes by PLA with DTM with barcodes identifying the targeted proteins at both ends. d) The reporter molecules are hybridized to oligonucleotides complementary to pairs of protein tags (e.g. N1N2), on a microarray, thereby allowing the reporter strands to be ligated into circles. e) The circularized reporter molecules finally template RCA, primed by the oligonucleo- tides on the array, and the RCA products are detected by hybridization with Cy3 or Cy5 labeled detection oligonucleotides. f) The Cy3 and Cy5 intensities are measured, and the ratios between the two colors are analyzed for each feature to detect differences in interaction patterns and protein abundances between the two samples. doi:10.1371/journal.pone.0040405.g001 shows that the assay is able to distinguish concentration differences of the investigated protein of less than two-fold over the full range. Next, we investigated if changes in protein concentration also could be measured in realistic biological samples, and for a greater number of targeted proteins. We prepared cell lysates from cells overexpressing members of the well-studied NFkB family of proteins, namely p50, RelA, RelB, and IkBa. The lysates were split in two aliquots and VEGF was spiked at a concentration of 10 pM into one of them. The pairs of samples were compared in an assay with PLA probes present for detection of all five proteins. Figure 2c presents the results in an MA-plot where the average intensity for a protein is plotted against the ratio between the two colors detected in the array. This illustrates the average of the protein concentration in both samples (A; x-axis) versus the difference in protein concentration between the two samples (M; y- axis), clearly showing that VEGF gives an average signal in the middle of the range of protein concentrations but that higher signals were detected for the sample with added VEGF as compared to the sample where no VEGF was spiked in. Accordingly, the assays distinctly revealed differences in VEGF concentrations between the two samples also in the complex context of cellular lysates. We used PLA with DTM readout for assessing pairwise protein interactions among the NFkB subunits RelA, RelB, and p50. It is well known that all these proteins can dimerize with each other both as homo- and heterodimers, and that the biological functions of these dimers can be prevented by forming larger complexes with the inhibitory IkB proteins (e.g. IkBa, reviewed in Hoffman et al. 2007 [24]). As a proof of concept we therefore investigated all six possible binary interactions between these four proteins. We also included reagents for detecting a housekeeping protein (glyceral- dehydes 3-phosphate dehydrogenase; GAPDH) in order to normalize for any differences in sample input, and we thus investigated ten possible binary interactions in total. As a first measure we developed an antibody validation pipeline (Figure S4) to ensure that the antibodies we used could recognize the target protein in native form also while in complex with its known interaction partners and in such a manner that each protein target could first be captured using an immobilized antibody, followed by the addition of two PLA probes. This was validated by (i) western blot analyses of lysates from cells transfected with the target protein in comparison to wild type lysates, (ii) Co-IP read out by western blot and (iii) SP-PLA with different combinations of antibodies targeting the same protein. Since SP-PLA requires the antigen to be captured and then bound by at least two PLA probes, in total at least three epitopes must be available for simultaneous binding on the same target molecule or pairs of molecules. Therefore, in some cases antibodies raised against different parts of the protein had to be combined to generate functional SP-PLAs. In the validation pipeline we have disregarded the fact that some of the target proteins may be present as homodimers. As a result of this we cannot tell to what extent the detection of protein content reflects the total protein concentration or that of homodimers. The specificity of the PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e40405