combination of target and ligand could be detected in the same in vitro experiment. In the present report we have extended this concept to the analysis of interacting proteins by using proximity ligation for detecting and measuring interacting proteins. The proximity ligation assay (PLA) [7] is an immunoassay utilizing so-called PLA probes – affinity reagents such as antibodies [8] modified with DNA oligonucleotides – for detecting and reporting the presence of proteins either in solution or in situ [9]. When two PLA probes bind the same or two interacting target molecules, the attached oligonucleotides are brought in close proximity. These oligonucleotides can then be joined by ligation to form an amplifiable reporter molecule. The requirement for recognition by two affinity reagents in proximity in order to generate a reporter molecule, followed by amplification by PCR or rolling-circle amplification (RCA), provides for highly sensitive assays to detect low amounts of proteins. The assays can also be used to study interactions among proteins [9–11] or between protein and DNA sequences [12,13]. PLA has been used to detect potential biomarkers in plasma, serum, cerebrospinal fluid, and cell lysates, both single proteins, protein aggregates (Ab proto- fibrils) [14], and interacting proteins have been targeted [10,11]. In situ PLA is a variant of the technique that permits visualization of the location of proteins, protein-protein interactions, and second- ary modifications such as phosphorylations [15] and glycosylations [16] in fixed cells and tissues without a need for genetic modification of the investigated cells. PLA has the potential to simultaneously assess large numbers of proteins and protein-protein interactions in a sample, since tag sequences can be introduced in the synthetic reporter DNA molecules, and several methods are available for detecting numerous DNA sequences simultaneously. Solution-phase PLA has been adapted for multiplex analyses of proteins with readout, either by quantitative PCR [17–19] or using next generation DNA sequencing [20]. Here we have combined PLA with readout via dual tag microarrays (DTM) [21] for parallel, sensitive detection of sets of proteins and protein-protein interactions. Using the DTM technique each microarray feature specifically detects and amplifies signals from reporter nucleic acid molecules that comprise two tag sequence elements, representing the interacting proteins. We have previously used the DTM technique to measure cDNA levels as well as reporter molecules from proximity ligation and padlock probe assays, achieving a radically decreased risk of cross-hybridization compared to standard microarray approaches [21]. Here we apply PLA with DTM readout to identify pairs of DNA tags, derived from the two PLA probes whose DNA strands have become joined by ligation upon coordinated binding to the same target protein or protein complex. The DTM readout can be applied for analysis of PLA products reflecting protein levels and interactions both in liquid samples and for fixed tissue sections on slides, to provide measures of all binary combinations of PLA probes. The approach can be generalized to interrogate all interactions within larger sets of investigated proteins. It has previously been shown that dual color microarray analysis of proteins, where two samples are compared directly against each other, outperforms single color approaches when it comes to reproducibility and power of discrimination of the assay [22]. We therefore designed a cassette connector oligonucleotide to facilitate sample barcoding for dual-color readout of results from analyses of pairs of samples. The combination of PLA with dual color DTM readout for multiplex detection of proteins and protein-protein interactions is illustrated in Figure 1. The assay is designed for direct comparison of each potential protein interaction in a set of investigated Profiling Protein Complexes by PLA with DTM proteins between two samples in the same spot of a microarray using dual color readout. PLA is performed either on immune- precipitates of molecules of interest from cell lysates, or on cells fixed on a microscope slide, using a variant of methods previously described [10,23] (Figure 1a). Reaction products from individual samples are barcoded with a unique DNA sequence by interposing a short sample-specific sequence when pairs of PLA probes are joined by ligation (Figure 1b). Thereafter the ligated reporter molecules from the two samples to be compared are pooled into one reaction tube and jointly amplified by PCR (Figure 1c). The amplified reporter molecules are allowed to hybridize to the arrays (Figure 1d). A ligase is subsequently added, allowing only the reporter with two barcode tags complementary to the array oligonucleotide to be ligated into circles. The circles will template localized DNA amplification by RCA on the microarray, as previously described [21]. Detection oligonucleotides, specific for the two sample barcodes and labeled with Cy3 or Cy5, are added (Figure 1e). The recorded fluorescence ratio of the two colors in each spot provides a measure of the difference in levels of proteins or protein interactions between the two samples (Figure 1f). To our knowledge this is the first description of an immunoassay capable of detecting all possible binary interactions among a given set of proteins in biological samples. We demonstrate analyses of protein interactions both in cell lysates and in cells fixed onto glass slides, with readout using binary microarrays. Results Proximity ligation using the SP-PLA technique provides opportunities to investigate interactions among proteins in cells and tissues. Here we have optimized the SP-PLA protocol for analyzing the levels of all pairwise protein interactions in a set of targeted proteins, with microarray readout as presented in the supplementary material (File S1, and Figure S1, Figure S2, Figure S3). To benchmark the protocol and to establish its performance for protein detection we first analyzed the presence of VEGF and IL8, spiked into buffer, before continuing to detect protein interactions in more complex samples. Proximity ligation was performed on two samples, labeling reaction products with sample-specific sequences. Products from pairs of samples were pooled prior to PCR and DTM readout, where products from the two samples were detected in the same array features using probes labeled with distinct fluorophores. The purpose of the experiment was to investigate differences in protein abundance between two samples in the presence of another protein maintained at the same level in all samples. The concentration of VEGF was varied over a broad range in order to investigate the dynamic range of the assay, while always maintaining a constant ten-fold different concentra- tion of VEGF between the two samples to be pairwise compared. The highest VEGF concentrations used were 1 nM/10 nM, and the lowest 10 fM/100 fM. In addition, all samples contained the reference protein IL8 at a constant concentration of 10 pM. Figure 2a presents the fluorescence ratio for VEGF normalized by the fluorescence ratio for IL8. The experiment demonstrates that the ten-fold difference in VEGF concentration between the two samples could be detected over a broad range of concentrations from ,10 nM down to ,100 fM, with IL8 as a constant reference in both samples. To next investigate the precision of the assay, two-fold dilutions of IL8 in PLA buffer were analyzed, along with 10 pM of VEGF spiked into each sample as PLA controls and for normalization. These samples were all individually compared with a sample of 10 pM IL8 and 10 pM VEGF spiked into PLA buffer. Figure 2b PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e40405