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SH2-PLA: a sensitive in-solution approach for quantification of modular domain binding by proximity ligation and real-time PCR.

Thompson CM, Bloom LR, Ogiue-Ikeda M, Machida K - BMC Biotechnol. (2015)

Bottom Line: If the GST-SH2 and EGFR are in close proximity as a result of SH2-phosphotyrosine interactions, the two oligonucleotides are brought within a suitable distance for ligation to occur, allowing for efficient complex amplification via real-time PCR.SH2 binding kinetics determined by PLA-SH2 showed good agreement with established far-Western analyses for A431 and Cos1 cells stimulated with EGF at various times and doses.Further, we showed that PLA-SH2 can survey lung cancer tissues using 1 μl lysate without requiring phospho-enrichment.

View Article: PubMed Central - PubMed

Affiliation: Raymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, Genetics and Genome Sciences, University of Connecticut School of Medicine, 400 Farmington Avenue, 06030, Farmington, CT, USA. thompsc1@mskcc.org.

ABSTRACT

Background: There is a great interest in studying phosphotyrosine dependent protein-protein interactions in tyrosine kinase pathways that play a critical role in many aspects of cellular function. We previously established SH2 profiling, a phosphoproteomic approach based on membrane binding assays that utilizes purified Src Homology 2 (SH2) domains as a molecular tool to profile the global tyrosine phosphorylation state of cells. However, in order to use this method to investigate SH2 binding sites on a specific target in cell lysate, additional procedures such as pull-down or immunoprecipitation which consume large amounts of sample are required.

Results: We have developed PLA-SH2, an alternative in-solution modular domain binding assay that takes advantage of Proximity Ligation Assay and real-time PCR. The SH2-PLA assay utilizes oligonucleotide-conjugated anti-GST and anti-EGFR antibodies recognizing a GST-SH2 probe and cellular EGFR, respectively. If the GST-SH2 and EGFR are in close proximity as a result of SH2-phosphotyrosine interactions, the two oligonucleotides are brought within a suitable distance for ligation to occur, allowing for efficient complex amplification via real-time PCR. The assay detected signal across at least 3 orders of magnitude of lysate input with a linear range spanning 1-2 orders and a low femtomole limit of detection for EGFR phosphotyrosine. SH2 binding kinetics determined by PLA-SH2 showed good agreement with established far-Western analyses for A431 and Cos1 cells stimulated with EGF at various times and doses. Further, we showed that PLA-SH2 can survey lung cancer tissues using 1 μl lysate without requiring phospho-enrichment.

Conclusions: We showed for the first time that interactions between SH2 domain probes and EGFR in cell lysate can be determined in a microliter-scale assay using SH2-PLA. The obvious benefit of this method is that the low sample requirement allows detection of SH2 binding in samples which are difficult to analyze using traditional protein interaction assays. This feature along with short assay runtime makes this method a useful platform for the development of high throughput assays to determine modular domain-ligand interactions which could have wide-ranging applications in both basic and translational cancer research.

No MeSH data available.


Related in: MedlinePlus

Estimation of EGFR phosphotyrosines at the limit of detection. To define an absolute lower limit of detection (LOD) for SH2-PLA, the total amount of EGFR phosphotyrosines in sample cell lysate was estimated using a phosphotyrosine standard sample and quantitative dot blotting analyses. a, Recombinant c-Abl protein, the pTyr-standard sample, was treated with tyrosine specific phosphatases PTP1B and TC-PTP. The amount of hydrolyzed phosphotyrosine was quantified by malachite green phosphatase assay (∆Abl PO43−). Left panel shows anti-Abl and anti-phosphotyrosine blots for phosphatase-treated (Abl PTP+) and -untreated (Abl PTP-) samples. After the PTP treatment, the level of c-Abl tyrosine phosphorylation was greatly reduced but weak phosphorylation was still detectable with longer exposure time. The right panel shows a plot of the phosphate standard used for the quantification. Red circle, untreated c-Abl; blue circle, PTP-treated c-Abl; yellow circles, the kit supplied phosphate standard. From this analysis, ∆Abl PO43 was estimated to be 5.7 pmol per μg of the c-Abl protein. b, Quantitative dot blotting. The total pTyr in the EGF-stimulated Cos1 cell lysate was estimated from a pTyr standard curve generated from anti-phosphotyrosine dot blotting. Upper panel shows raw anti-Abl and anti-pTyr blots. Serially diluted c-Abl pTyr standard (left to right 3.1–0.02 ng per spot) and 0.01 μg EGF-stimulated Cos1 samples were spotted on nitrocellulose membrane (performed in triplicate). The middle panel shows the resulting pTyr standard plot with the quantified signal intensities. The pTyr amount in the EGF-stimulated Cos1 lysate was estimated to be 0.08 pmol per μg lysate. Subsequently, an anti-pTyr Western analysis for A431 and Cos1 samples was performed, relative intensities of the EGFR bands were calculated, and the amount of EGFR pTyr in the EGF-stimulated A431 sample was estimated to be 0.122 pmol/μg. Thus 2 ng of EGF-stimulated A431 sample, which is the lower limit for SH2-PLA detection, would contain 0.243 femtomole EGFR pTyr. See Methods and Additional file 1: Figure S4 for more information
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Fig3: Estimation of EGFR phosphotyrosines at the limit of detection. To define an absolute lower limit of detection (LOD) for SH2-PLA, the total amount of EGFR phosphotyrosines in sample cell lysate was estimated using a phosphotyrosine standard sample and quantitative dot blotting analyses. a, Recombinant c-Abl protein, the pTyr-standard sample, was treated with tyrosine specific phosphatases PTP1B and TC-PTP. The amount of hydrolyzed phosphotyrosine was quantified by malachite green phosphatase assay (∆Abl PO43−). Left panel shows anti-Abl and anti-phosphotyrosine blots for phosphatase-treated (Abl PTP+) and -untreated (Abl PTP-) samples. After the PTP treatment, the level of c-Abl tyrosine phosphorylation was greatly reduced but weak phosphorylation was still detectable with longer exposure time. The right panel shows a plot of the phosphate standard used for the quantification. Red circle, untreated c-Abl; blue circle, PTP-treated c-Abl; yellow circles, the kit supplied phosphate standard. From this analysis, ∆Abl PO43 was estimated to be 5.7 pmol per μg of the c-Abl protein. b, Quantitative dot blotting. The total pTyr in the EGF-stimulated Cos1 cell lysate was estimated from a pTyr standard curve generated from anti-phosphotyrosine dot blotting. Upper panel shows raw anti-Abl and anti-pTyr blots. Serially diluted c-Abl pTyr standard (left to right 3.1–0.02 ng per spot) and 0.01 μg EGF-stimulated Cos1 samples were spotted on nitrocellulose membrane (performed in triplicate). The middle panel shows the resulting pTyr standard plot with the quantified signal intensities. The pTyr amount in the EGF-stimulated Cos1 lysate was estimated to be 0.08 pmol per μg lysate. Subsequently, an anti-pTyr Western analysis for A431 and Cos1 samples was performed, relative intensities of the EGFR bands were calculated, and the amount of EGFR pTyr in the EGF-stimulated A431 sample was estimated to be 0.122 pmol/μg. Thus 2 ng of EGF-stimulated A431 sample, which is the lower limit for SH2-PLA detection, would contain 0.243 femtomole EGFR pTyr. See Methods and Additional file 1: Figure S4 for more information

Mentions: First, using a baculovirus expression system, we generated recombinant GST fused c-Abl protein to serve as the standard for phosphotyrosine. Then we treated the Abl protein with the tyrosine specific phosphatases PTP1B and TC-PTP. Anti-phosphotyrosine blots of treated and untreated Abl proteins indicated that phosphorylated Abl protein was mostly dephosphorylated by phosphatase treatment. Following the treatment, we quantified the amount of free phosphate, which is the hydrolyzed product of phosphotyrosine, using a phosphate standard curve generated with a malachite green phosphatase assay (∆Abl PO43− = 5.7 pmol/μg, Fig. 3a).Fig. 3


SH2-PLA: a sensitive in-solution approach for quantification of modular domain binding by proximity ligation and real-time PCR.

Thompson CM, Bloom LR, Ogiue-Ikeda M, Machida K - BMC Biotechnol. (2015)

Estimation of EGFR phosphotyrosines at the limit of detection. To define an absolute lower limit of detection (LOD) for SH2-PLA, the total amount of EGFR phosphotyrosines in sample cell lysate was estimated using a phosphotyrosine standard sample and quantitative dot blotting analyses. a, Recombinant c-Abl protein, the pTyr-standard sample, was treated with tyrosine specific phosphatases PTP1B and TC-PTP. The amount of hydrolyzed phosphotyrosine was quantified by malachite green phosphatase assay (∆Abl PO43−). Left panel shows anti-Abl and anti-phosphotyrosine blots for phosphatase-treated (Abl PTP+) and -untreated (Abl PTP-) samples. After the PTP treatment, the level of c-Abl tyrosine phosphorylation was greatly reduced but weak phosphorylation was still detectable with longer exposure time. The right panel shows a plot of the phosphate standard used for the quantification. Red circle, untreated c-Abl; blue circle, PTP-treated c-Abl; yellow circles, the kit supplied phosphate standard. From this analysis, ∆Abl PO43 was estimated to be 5.7 pmol per μg of the c-Abl protein. b, Quantitative dot blotting. The total pTyr in the EGF-stimulated Cos1 cell lysate was estimated from a pTyr standard curve generated from anti-phosphotyrosine dot blotting. Upper panel shows raw anti-Abl and anti-pTyr blots. Serially diluted c-Abl pTyr standard (left to right 3.1–0.02 ng per spot) and 0.01 μg EGF-stimulated Cos1 samples were spotted on nitrocellulose membrane (performed in triplicate). The middle panel shows the resulting pTyr standard plot with the quantified signal intensities. The pTyr amount in the EGF-stimulated Cos1 lysate was estimated to be 0.08 pmol per μg lysate. Subsequently, an anti-pTyr Western analysis for A431 and Cos1 samples was performed, relative intensities of the EGFR bands were calculated, and the amount of EGFR pTyr in the EGF-stimulated A431 sample was estimated to be 0.122 pmol/μg. Thus 2 ng of EGF-stimulated A431 sample, which is the lower limit for SH2-PLA detection, would contain 0.243 femtomole EGFR pTyr. See Methods and Additional file 1: Figure S4 for more information
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig3: Estimation of EGFR phosphotyrosines at the limit of detection. To define an absolute lower limit of detection (LOD) for SH2-PLA, the total amount of EGFR phosphotyrosines in sample cell lysate was estimated using a phosphotyrosine standard sample and quantitative dot blotting analyses. a, Recombinant c-Abl protein, the pTyr-standard sample, was treated with tyrosine specific phosphatases PTP1B and TC-PTP. The amount of hydrolyzed phosphotyrosine was quantified by malachite green phosphatase assay (∆Abl PO43−). Left panel shows anti-Abl and anti-phosphotyrosine blots for phosphatase-treated (Abl PTP+) and -untreated (Abl PTP-) samples. After the PTP treatment, the level of c-Abl tyrosine phosphorylation was greatly reduced but weak phosphorylation was still detectable with longer exposure time. The right panel shows a plot of the phosphate standard used for the quantification. Red circle, untreated c-Abl; blue circle, PTP-treated c-Abl; yellow circles, the kit supplied phosphate standard. From this analysis, ∆Abl PO43 was estimated to be 5.7 pmol per μg of the c-Abl protein. b, Quantitative dot blotting. The total pTyr in the EGF-stimulated Cos1 cell lysate was estimated from a pTyr standard curve generated from anti-phosphotyrosine dot blotting. Upper panel shows raw anti-Abl and anti-pTyr blots. Serially diluted c-Abl pTyr standard (left to right 3.1–0.02 ng per spot) and 0.01 μg EGF-stimulated Cos1 samples were spotted on nitrocellulose membrane (performed in triplicate). The middle panel shows the resulting pTyr standard plot with the quantified signal intensities. The pTyr amount in the EGF-stimulated Cos1 lysate was estimated to be 0.08 pmol per μg lysate. Subsequently, an anti-pTyr Western analysis for A431 and Cos1 samples was performed, relative intensities of the EGFR bands were calculated, and the amount of EGFR pTyr in the EGF-stimulated A431 sample was estimated to be 0.122 pmol/μg. Thus 2 ng of EGF-stimulated A431 sample, which is the lower limit for SH2-PLA detection, would contain 0.243 femtomole EGFR pTyr. See Methods and Additional file 1: Figure S4 for more information
Mentions: First, using a baculovirus expression system, we generated recombinant GST fused c-Abl protein to serve as the standard for phosphotyrosine. Then we treated the Abl protein with the tyrosine specific phosphatases PTP1B and TC-PTP. Anti-phosphotyrosine blots of treated and untreated Abl proteins indicated that phosphorylated Abl protein was mostly dephosphorylated by phosphatase treatment. Following the treatment, we quantified the amount of free phosphate, which is the hydrolyzed product of phosphotyrosine, using a phosphate standard curve generated with a malachite green phosphatase assay (∆Abl PO43− = 5.7 pmol/μg, Fig. 3a).Fig. 3

Bottom Line: If the GST-SH2 and EGFR are in close proximity as a result of SH2-phosphotyrosine interactions, the two oligonucleotides are brought within a suitable distance for ligation to occur, allowing for efficient complex amplification via real-time PCR.SH2 binding kinetics determined by PLA-SH2 showed good agreement with established far-Western analyses for A431 and Cos1 cells stimulated with EGF at various times and doses.Further, we showed that PLA-SH2 can survey lung cancer tissues using 1 μl lysate without requiring phospho-enrichment.

View Article: PubMed Central - PubMed

Affiliation: Raymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, Genetics and Genome Sciences, University of Connecticut School of Medicine, 400 Farmington Avenue, 06030, Farmington, CT, USA. thompsc1@mskcc.org.

ABSTRACT

Background: There is a great interest in studying phosphotyrosine dependent protein-protein interactions in tyrosine kinase pathways that play a critical role in many aspects of cellular function. We previously established SH2 profiling, a phosphoproteomic approach based on membrane binding assays that utilizes purified Src Homology 2 (SH2) domains as a molecular tool to profile the global tyrosine phosphorylation state of cells. However, in order to use this method to investigate SH2 binding sites on a specific target in cell lysate, additional procedures such as pull-down or immunoprecipitation which consume large amounts of sample are required.

Results: We have developed PLA-SH2, an alternative in-solution modular domain binding assay that takes advantage of Proximity Ligation Assay and real-time PCR. The SH2-PLA assay utilizes oligonucleotide-conjugated anti-GST and anti-EGFR antibodies recognizing a GST-SH2 probe and cellular EGFR, respectively. If the GST-SH2 and EGFR are in close proximity as a result of SH2-phosphotyrosine interactions, the two oligonucleotides are brought within a suitable distance for ligation to occur, allowing for efficient complex amplification via real-time PCR. The assay detected signal across at least 3 orders of magnitude of lysate input with a linear range spanning 1-2 orders and a low femtomole limit of detection for EGFR phosphotyrosine. SH2 binding kinetics determined by PLA-SH2 showed good agreement with established far-Western analyses for A431 and Cos1 cells stimulated with EGF at various times and doses. Further, we showed that PLA-SH2 can survey lung cancer tissues using 1 μl lysate without requiring phospho-enrichment.

Conclusions: We showed for the first time that interactions between SH2 domain probes and EGFR in cell lysate can be determined in a microliter-scale assay using SH2-PLA. The obvious benefit of this method is that the low sample requirement allows detection of SH2 binding in samples which are difficult to analyze using traditional protein interaction assays. This feature along with short assay runtime makes this method a useful platform for the development of high throughput assays to determine modular domain-ligand interactions which could have wide-ranging applications in both basic and translational cancer research.

No MeSH data available.


Related in: MedlinePlus