Sensitive protein detection via triple-binder proximity ligation assays

Schallmeiner, Edith; Oksanen, Elli; Ericsson, Olle; Spångberg, Lena; Eriksson, Susann; Stenman, Ulf‐Håkan; Pettersson, Kim; Landegren, Ulf

doi:10.1038/nmeth974

Cited by 120 publications

(106 citation statements)

References 13 publications

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“…We have previously demonstrated that the proximity ligation mechanism can be extended to using sets of three antibodies (9,20). This could provide unique opportunities to investigate constellations of several phosphorylations or other PTMs in individual protein molecules, something that would present great difficulties using methods like mass spectrometry.…”

Section: Discussionmentioning

confidence: 99%

In Situ Detection of Phosphorylated Platelet-derived Growth Factor Receptor β Using a Generalized Proximity Ligation Method

Jarvius

Paulsson

Weibrecht

et al. 2007

Molecular & Cellular Proteomics

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205

177

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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 ␤ (PDGFR␤) 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 PDGFR␤ in human embryonic kidney cells stably overexpressing human influenza hemagglutinin-tagged human PDGFR␤ in porcine aortic endothelial cells transfected with the ␤-receptor, but not in cells transfected with the ␣-receptor, and also in immortalized human foreskin fibroblasts, BJ hTert, endogenously expressing the PDGFR␤. We furthermore visualized tyrosine phosphorylated PDGFR␤ 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. Molecular & Cellular Proteomics 6:1500 -1509, 2007.

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Section: Discussionmentioning

confidence: 99%

In Situ Detection of Phosphorylated Platelet-derived Growth Factor Receptor β Using a Generalized Proximity Ligation Method

Jarvius

Paulsson

Weibrecht

et al. 2007

Molecular & Cellular Proteomics

Self Cite

205

177

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“…In asymmetric PLA, connector oligonucleotide binding affinity towards the probe strands is weakened in one end, reducing the probability of target-independent ligation (29). Triple-specific proximity ligation assay (3PLA) lowers background signal by using weakly hybridizing oligonucleotides to temporarily block unbound probes and increases specificity through triple recognition of three separate target probes (30). Landegren et al reported a 100-fold increase in sensitivity compared to standard homogenous PLA when assaying recombinant VEGF (22).…”

Section: Methodological Considerationsmentioning

confidence: 99%

Sensitive Ligand-Based Protein Quantification Using Immuno-PCR: A Critical Review Of Single-Probe And Proximity Ligation Assays

et al. 2014

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Quantitative PCR (qPCR) of reverse-transcribed mRNA has revolutionized gene expression analyses. qPCR analysis is based on the prevalent assumption that mRNA transcript numbers provide an adequate measure of specific biomarker expression. However, taking the complexity of protein turnover into account, there is a need to correlate qPCR-derived transcriptional patterns with protein translational patterns so as to not leave behind important pathobiological details. One emerging approach in protein analysis is PCR-coupled protein quantification, often denoted as immuno-PCR (iPCR), which targets soluble proteins. Here we review recent trends and applications in iPCR assays that may bridge the gap between classical enzyme-linked immunosorbent assays and mass spectrometry methodologies in terms of sensitivity and multiplexing.

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“…PDGF-BB was measured in lysates and medium from four plates for each genotype. Measurements were done in triplicate using triple proximity ligation assay as described (Schallmeiner et al 2007). …”

Section: Pdgf-bb Proximity Ligation Assaymentioning

confidence: 99%

DefectiveN-sulfation of heparan sulfate proteoglycans limits PDGF-BB binding and pericyte recruitment in vascular development

Abramsson¹,

Kurup²,

Busse³

et al. 2007

Genes Dev.

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145

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During vascular development, endothelial platelet-derived growth factor B (PDGF-B) is critical for pericyte recruitment. Deletion of the conserved C-terminal heparin-binding motif impairs PDGF-BB retention and pericyte recruitment in vivo, suggesting a potential role for heparan sulfate (HS) in PDGF-BB function during vascular development.We studied the participation of HS chains in pericyte recruitment using two mouse models with altered HS biosynthesis. Reduction of N-sulfation due to deficiency in N-deacetylase/ N-sulfotransferase-1 attenuated PDGF-BB binding in vitro, and led to pericyte detachment and delayed pericyte migration in vivo. Reduced N-sulfation also impaired PDGF-BB signaling and directed cell migration, but not proliferation. In contrast, HS from glucuronyl C5-epimerase mutants, which is extensively N-and 6-O-sulfated, but lacks 2-O-sulfated L-iduronic acid residues, retained PDGF-BB in vitro, and pericyte recruitment in vivo was only transiently delayed. These observations were supported by in vitro characterization of the structural features in HS important for PDGF-BB binding. We conclude that pericyte recruitment requires HS with sufficiently extended and appropriately spaced N-sulfated domains to retain PDGF-BB and activate PDGF receptor ␤ (PDGFR␤) signaling, whereas the detailed sequence of monosaccharide and sulfate residues does not appear to be important for this interaction.[Keywords: PDGF-B; angiogenesis; heparan sulfate; pericyte; vascular development] Supplemental material is available at http://www.genesdev.org. Tissue morphogenesis depends on cell-cell interactions, controlling directed cell migration proliferation, differentiation, and cell survival. Specificity is often regulated at the level of selective ligand-receptor interaction. However, the spatial distribution and local concentration of the ligand determine the range of the signal, and, as exemplified by morphogens of the hedgehog, TGF␤, and Wnt family members, also the nature of the signal. Indeed, spatial restriction defines the activities of most peptide growth factors and many secreted neural guidance molecules. In vascular development, peptide growth factors of the VEGF and platelet-derived growth factor (PDGF) families regulate the migration and proliferation of endothelial cells and supporting mural cells; i.e., pericytes (PC) and vascular smooth muscle cells (vSMC). The longitudinal migration and proliferation of vSMC/PC depend on paracrine signaling of endothelial derived PDGF-B to PDGF receptor-␤ (PDGFR␤) expressed on vSMC/PC (Lindahl et al. 1997;Hellström et al. 1999). PDGF-B is secreted as a homodimer (PDGF-BB), which signals by mediating dimerization of its receptor. Conditional inactivation of Pdgf-b in the endothelium demonstrated that endothelial cells are the

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Sensitive protein detection via triple-binder proximity ligation assays

Cited by 120 publications

References 13 publications

In Situ Detection of Phosphorylated Platelet-derived Growth Factor Receptor β Using a Generalized Proximity Ligation Method

In Situ Detection of Phosphorylated Platelet-derived Growth Factor Receptor β Using a Generalized Proximity Ligation Method

Sensitive Ligand-Based Protein Quantification Using Immuno-PCR: A Critical Review Of Single-Probe And Proximity Ligation Assays

DefectiveN-sulfation of heparan sulfate proteoglycans limits PDGF-BB binding and pericyte recruitment in vascular development

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