Western blotting via proximity ligation for high performance protein analysis

Western blotting is a powerful and widely used method, but limitations in detection sensitivity and specificity, and dependence upon high quality antibodies to detect targeted proteins, are hurdles to overcome. The in situ proximity ligation assay, based on dual antibody recognition and powerful localized signal amplification, offers increased detection sensitivity and specificity, along with an ability to identify complex targets such as phosphorylated or interacting proteins. Here we have applied the in situ proximity ligation assay mechanism in Western blotting. This combination allowed the use of isothermal rolling circle amplification of DNA molecules formed in target-specific ligation reaction, for 16-fold or greater increase in detection sensitivity. The increased specificity because of dual antibody recognition ensured highly selective assays, detecting the specific band when combinations of two cross-reactive antitubulin antibodies were used (i.e. both producing distinct nonspecific bands in traditional Western blotting). We also demonstrated detection of phosphorylated platelet-derived growth factor receptor β by proximity ligation with one antibody directed against the receptor and another directed against the phosphorylated tyrosine residue. This avoided the need for stripping and re-probing the membrane or aligning two separate traditional blots. We demonstrate that the high-performance in situ proximity ligation-based Western blotting described herein is compatible with detection via enhanced chemiluminescence and fluorescence detection systems, and can thus be readily employed in any laboratory.     

Sensitive protein detection via triple-binder proximity ligation assays

The detection of weakly expressed proteins and protein complexes in biological samples represents a fundamental challenge. We have developed a new proximity-ligation strategy named 3PLA that uses three recognition events for the highly specific and sensitive detection of as little as a hundred molecules of the vascular endothelial growth factor (VEGF), the biomarkers troponin I, and prostate-specific antigen (PSA) alone or in complex with an inhibitor--demonstrating the versatility of 3PLA.     

Sensitive detection of aggregated prion protein via proximity ligation

The DNA assisted solid-phase proximity ligation assay (SP-PLA) provides a unique opportunity to specifically detect prion protein (PrP) aggregates by investigating the collocation of 3 or more copies of the specific protein. We have developed an SP-PLA that can detect PrP aggregates in brain homogenates from infected hamsters even after a 10⁷-fold dilution. In contrast, brain homogenate from uninfected animals did not generate a detectable signal at 100-fold higher concentration. Using either of the 2 monoclonal anti-PrP antibodies, 3F4 and 6H4, we successfully detected low concentrations of aggregated PrP. The presented results provide a proof of concept that this method might be an interesting tool in the development of diagnostic approaches of prion diseases.     

A sense of closeness: protein detection by proximity ligation

Highly specific and sensitive procedures will be required to evaluate proteomes. Proximity ligation is a recently introduced mechanism for protein analysis. In this technique, the convergence of sets of protein-binding reagents on individual target molecules juxtaposes attached nucleic acid sequences. Through a ligation reaction a DNA reporter sequence is created, which can be amplified. The procedure thus encodes detected proteins as specific nucleic acid sequences in what may be viewed as a reverse translation reaction.     

Multiplexed protein detection by proximity ligation for cancer biomarker validation

We present a proximity ligation-based multiplexed protein detection procedure in which several selected proteins can be detected via unique nucleic-acid identifiers and subsequently quantified by real-time PCR. The assay requires a 1-microl sample, has low-femtomolar sensitivity as well as five-log linear range and allows for modular multiplexing without cross-reactivity. The procedure can use a single polyclonal antibody batch for each target protein, simplifying affinity-reagent creation for new biomarker candidates.     

Self-assembly of proximity probes for flexible and modular proximity ligation assays

Proximity ligation assay (PLA) is a recently developed strategy for protein analysis in which antibody-based detection of a target protein via a DNA ligation reaction of oligonucleotides linked to the antibodies results in the formation of an amplifiable DNA strand suitable for analysis. Here we describe a faster and more cost-effective strategy to construct the antibody-based proximity ligation probes used in PLA that is based on the noncovalent interaction of biotinylated oligonucleotides with streptavidin followed by the interaction of this complex with biotinylated antibodies.     

Expressed protein ligation. Method and applications.

The introduction of noncanonical amino acids and biophysical probes into peptides and proteins, and total or segmental isotopic labelling has the potential to greatly aid the determination of protein structure, function and protein-protein interactions. To obtain a peptide as large as possible by solid-phase peptide synthesis, native chemical ligation was introduced to enable synthesis of proteins of up to 120 amino acids in length. After the discovery of inteins, with their self-splicing properties and their application in protein synthesis, the semisynthetic methodology, expressed protein ligation, was developed to circumvent size limitation problems. Today, diverse expression vectors are available that allow the production of N- and C-terminal fragments that are needed for ligation to produce large amounts and high purity protein(s) (protein alpha-thioesters and peptides or proteins with N-terminal Cys). Unfortunately, expressed protein ligation is still limited mainly by the requirement of a Cys residue. Of course, additional Cys residues can be introduced into the sequence by site directed mutagenesis or synthesis, however, those mutations may disturb protein structure and function. Recently, alternative ligation approaches have been developed that do not require Cys residues. Accordingly, it is theoretically possible to obtain each modified protein using ligation strategies.     

Profiling cellular protein complexes by proximity ligation with dual tag microarray readout

Patterns of protein interactions provide important insights in basic biology, and their analysis plays an increasing role in drug development and diagnostics of disease. We have established a scalable technique to compare two biological samples for the levels of all pairwise interactions among a set of targeted protein molecules. The technique is a combination of the proximity ligation assay with readout via dual tag microarrays. In the proximity ligation assay protein identities are encoded as DNA sequences by attaching DNA oligonucleotides to antibodies directed against the proteins of interest. Upon binding by pairs of antibodies to proteins present in the same molecular complexes, ligation reactions give rise to reporter DNA molecules that contain the combined sequence information from the two DNA strands. The ligation reactions also serve to incorporate a sample barcode in the reporter molecules to allow for direct comparison between pairs of samples. The samples are evaluated using a dual tag microarray where information is decoded, revealing which pairs of tags that have become joined. As a proof-of-concept we demonstrate that this approach can be used to detect a set of five proteins and their pairwise interactions both in cellular lysates and in fixed tissue culture cells. This paper provides a general strategy to analyze the extent of any pairwise interactions in large sets of molecules by decoding reporter DNA strands that identify the interacting molecules.     

Multiplexed homogeneous proximity ligation assays for high-throughput protein biomarker research in serological material

A high throughput protein biomarker discovery tool has been developed based on multiplexed proximity ligation assays in a homogeneous format in the sense of no washing steps. The platform consists of four 24-plex panels profiling 74 putative biomarkers with sub-pm sensitivity each consuming only 1 μl of human plasma sample. The system uses either matched monoclonal antibody pairs or the more readily available single batches of affinity purified polyclonal antibodies to generate the target specific reagents by covalently linking with unique nucleic acid sequences. These paired sequences are united by DNA ligation upon simultaneous target binding forming a PCR amplicon. Multiplex proximity ligation assays thereby converts multiple target analytes into real-time PCR amplicons that are individually quantified using microfluidic high capacity qPCR in nano liter volumes. The assay shows excellent specificity, even in multiplex, by its dual recognition feature, its proximity requirement, and most importantly by using unique sequence specific reporter fragments on both antibody-based probes. To illustrate the potential of this protein detection technology, a pilot biomarker research project was performed using biobanked plasma samples for the detection of colorectal cancer using a multivariate signature.     

Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay

The activity of proteins is typically regulated by secondary modifications and by interactions with other partners, resulting in the formation of protein complexes whose functions depend on the participating proteins. Accordingly, it is of central importance to monitor the presence of interaction complexes as well as their localization, thus providing information about the types of cells where the proteins are located and in what sub-cellular compartment these interactions occur. Several methods for visualizing protein interactions in situ have been developed during the last decade. These methods in most cases involve genetic constructs, and they have been successfully used in assays of living cell maintained in tissue culture, but they cannot easily be implemented in studies of clinical specimens. For such samples, affinity reagents like antibodies can be used to target the interacting proteins. In this review we will describe the in situ proximity ligation assays (in situ PLA), a method that is suitable for visualizing protein interactions in both tissue sections and in vitro cell lines, and we discuss research tasks when this or other method may be selected.     

Studies of ion channels using expressed protein ligation

Expressed protein ligation (EPL) is a semisynthetic technique for the chemoselective ligation of a synthetic peptide to a recombinant peptide that results in a native peptide bond at the ligation site. EPL therefore allows us to engineer proteins with chemically defined, site-specific modifications. While EPL has been used mainly in investigations of soluble proteins, in recent years it has been increasingly used in investigations of integral membrane proteins. These include studies on the KcsA K(+) channel, the non-selective cation channel NaK, and the porin OmpF. These studies are discussed in this review.     

In situ detection of phosphorylated platelet-derived growth factor receptor beta using a generalized proximity ligation method

Improved methods are needed for in situ characterization of post-translational modifications in cell lines and tissues. For example, it is desirable to monitor the phosphorylation status of individual receptor tyrosine kinases in samples from human tumors treated with inhibitors to evaluate therapeutic responses. Unfortunately the leading methods for observing the dynamics of tissue post-translational modifications in situ, immunohistochemistry and immunofluorescence, exhibit limited sensitivity and selectivity. Proximity ligation assay is a novel method that offers improved selectivity through the requirement of dual recognition and increased sensitivity by including DNA amplification as a component of detection of the target molecule. Here we therefore established a generalized in situ proximity ligation assay to investigate phosphorylation of platelet-derived growth factor receptor beta (PDGFRbeta) in cells stimulated with platelet-derived growth factor BB. Antibodies specific for immunoglobulins from different species, modified by attachment of DNA strands, were used as secondary proximity probes together with a pair of primary antibodies from the corresponding species. Dual recognition of receptors and phosphorylated sites by the primary antibodies in combination with the secondary proximity probes was used to generate circular DNA strands; this was followed by signal amplification by replicating the DNA circles via rolling circle amplification. We detected tyrosine phosphorylated PDGFRbeta in human embryonic kidney cells stably overexpressing human influenza hemagglutinin-tagged human PDGFRbeta in porcine aortic endothelial cells transfected with the beta-receptor, but not in cells transfected with the alpha-receptor, and also in immortalized human foreskin fibroblasts, BJ hTert, endogenously expressing the PDGFRbeta. We furthermore visualized tyrosine phosphorylated PDGFRbeta in tissue sections from fresh frozen human scar tissue undergoing wound healing. The method should be of great value to study signal transduction, screen for effects of pharmacological agents, and enhance the diagnostic potential in histopathology.     

Comparative analysis of fluorescence resonance energy transfer (FRET) and proximity ligation assay (PLA)

Both fluorescence resonance energy transfer (FRET) and proximity ligation assay (PLA) are techniques used in the investigation of protein interactions but the latter has not been evaluated in a systematic way, prompting us to compare their performance quantitatively. Proteins were labeled with oligonucleotide- or fluorophore-conjugated antibodies and their proximity was analyzed by flow cytometry in order to obtain statistically robust data. Both intermolecular and intramolecular PLA signals reached saturation at high expression levels. At the same time, the FRET efficiency was independent of, while the FRET signal exhibited a strict linear correlation with the expression levels of proteins. When the density of oligonucleotide- and fluorophore-conjugated antibodies was systematically changed by competition with unlabeled antibodies the FRET signal was linearly proportional to the amount of bound fluorophore-tagged antibodies, whereas the PLA signal was again saturated. The saturation phenomenon in PLA could not be eliminated by decreasing the duration of the rolling circle amplification reaction. Our data imply that PLA is a semiquantitative measure of protein colocalizations due to non-linear effects in the reaction and that caution should be exercised when interpreting PLA data in a quantitative way.     

Quantification of siRNA using competitive qPCR

We have developed a PCR-based short interfering RNA (siRNA) quantification method based on competition between siRNA and a homologous DNA primer for annealing to template DNA, avoiding the requirement for prior conversion of RNA to cDNA. Primers and probe were designed to amplify regions of the human papillomavirus E6 or enhanced green fluorescent protein genes. Having confirmed siRNA could not act as primer for amplicon generation, the lowest competing primer concentration yielding a linear relationship between template DNA amount (0.1–50 ng) and cycle of threshold (Ct) was determined (6.25 nM). Under these conditions addition of sequence-specific siRNA to the competitive quantitative PCR (cqPCR), resulted in a dose-dependent linear increase in Ct value. 2′-O-methyl ribose-modified siRNA retained an ability to inhibit template amplification in serum, unlike unmodified siRNAs that were susceptible to endonucleases. Mismatch-bearing or truncated siRNAs failed to inhibit template amplification confirming sequence specificity and an ability to discriminate between degraded and non-degraded siRNA sequences. Following delivery of E6 siRNA to C33-A cells using oligofectamine or His6 reducible polymers, siRNA uptake was quantified by cqPCR, revealing dose-dependent uptake. We anticipate that cqPCR will allow accurate determination of siRNA pharmacokinetics following in vivo delivery, greatly facilitating development of therapeutic siRNA delivery strategies.     

ProteinSeq: high-performance proteomic analyses by proximity ligation and next generation sequencing

Despite intense interest, methods that provide enhanced sensitivity and specificity in parallel measurements of candidate protein biomarkers in numerous samples have been lacking. We present herein a multiplex proximity ligation assay with readout via realtime PCR or DNA sequencing (ProteinSeq). We demonstrate improved sensitivity over conventional sandwich assays for simultaneous analysis of sets of 35 proteins in 5 μl of blood plasma. Importantly, we observe a minimal tendency to increased background with multiplexing, compared to a sandwich assay, suggesting that higher levels of multiplexing are possible. We used ProteinSeq to analyze proteins in plasma samples from cardiovascular disease (CVD) patient cohorts and matched controls. Three proteins, namely P-selectin, Cystatin-B and Kallikrein-6, were identified as putative diagnostic biomarkers for CVD. The latter two have not been previously reported in the literature and their potential roles must be validated in larger patient cohorts. We conclude that ProteinSeq is promising for screening large numbers of proteins and samples while the technology can provide a much-needed platform for validation of diagnostic markers in biobank samples and in clinical use.     

Fast and reliable analysis of molecular motion using proximity relations and dimensionality reduction

The analysis of molecular motion starting from extensive sampling of molecular configurations remains an important and challenging task in computational biology. Existing methods require a significant amount of time to extract the most relevant motion information from such data sets. In this work, we provide a practical tool for molecular motion analysis. The proposed method builds upon the recent ScIMAP (Scalable Isomap) method, which, by using proximity relations and dimensionality reduction, has been shown to reliably extract from simulation data a few parameters that capture the main, linear and/or nonlinear, modes of motion of a molecular system. The results we present in the context of protein folding reveal that the proposed method characterizes the folding process essentially as well as ScIMAP. At the same time, by projecting the simulation data and computing proximity relations in a low-dimensional Euclidean space, it renders such analysis computationally practical. In many instances, the proposed method reduces the computational cost from several CPU months to just a few CPU hours, making it possible to analyze extensive simulation data in a matter of a few hours using only a single processor. These results establish the proposed method as a reliable and practical tool for analyzing motions of considerably large molecular systems and proteins with complex folding mechanisms.     

Expanding the scope of site-specific protein biotinylation strategies using small molecules

We present a new approach to site-specifically biotinylate protein in a cell-free protein synthesis system with puromycin-containing small molecules. With this new method, biotinylated proteins were generated from the DNA templates in a matter of hours, making it useful for protein microarray generation. We also validated that the method is compatible with other high-throughput cloning/proteomics methods.