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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

Applications of SH2-PLA assay. a, Practical limit of detection from cell culture. Two-fold serial dilutions of A431 cells were seeded to wells in a 96-well plate. Image series shows various 10x magnifications of diluted A431 cells with cell number indicated. Vav2 SH2:pEGFR interaction of starved or EGF-stimulated cell lysates was quantified by the SH2-PLA assay to resolve the assay detection limit. The Ct values from real-time PCR are shown with approximate numbers of cells per culture well or per assay (in brackets) underneath the chart. b, Time course and dose response of EGF stimulation. A431 and Cos1 cells were starved and stimulated with EGF at various times and concentrations as indicated. Upper panel shows far-Western blotting with Grb2 SH2 (25 μg lysate loaded per lane) and control blotting with anti-actin. Bottom panel shows Ct values of comparable SH2-PLA experiments loading 0.4 μg lysate per assay well. c, Correlation between far-Western and SH2-PLA assay. Using experimental results shown in B, EGFR band intensities of far-Western blot (X-axis) and average Ct values of SH2-PLA (Y-axis) in panel B were plotted and showed strong correlation. a.u., arbitrary unit; r, Pearson correlation coefficient. d, Application of SH2-PLA for cancer tissue analysis. The SH2-PLA/Western/far-Western analyses were performed using 10 lung cancer tissue samples. Upper panel shows Western and far-Western results with antibody/probe names indicated on the left. Only one sample (#3) shows an EGFR size band which also overlapped with bands detected by anti-pTyr and Grb2 SH2 (far-Western image represents 60-min exposure). The tyrosine phosphorylation level of the band is similar to the weak phosphorylation of EGFR in unstimulated A431 cells (right panel). The PLA-SH2 results for the same set of lung cancer samples are shown on the bottom. Consistent with the Grb2 far-Western result, only sample #3 had significant signal beyond the no protein control (NPC). The BG line indicates the background Ct value
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Fig4: Applications of SH2-PLA assay. a, Practical limit of detection from cell culture. Two-fold serial dilutions of A431 cells were seeded to wells in a 96-well plate. Image series shows various 10x magnifications of diluted A431 cells with cell number indicated. Vav2 SH2:pEGFR interaction of starved or EGF-stimulated cell lysates was quantified by the SH2-PLA assay to resolve the assay detection limit. The Ct values from real-time PCR are shown with approximate numbers of cells per culture well or per assay (in brackets) underneath the chart. b, Time course and dose response of EGF stimulation. A431 and Cos1 cells were starved and stimulated with EGF at various times and concentrations as indicated. Upper panel shows far-Western blotting with Grb2 SH2 (25 μg lysate loaded per lane) and control blotting with anti-actin. Bottom panel shows Ct values of comparable SH2-PLA experiments loading 0.4 μg lysate per assay well. c, Correlation between far-Western and SH2-PLA assay. Using experimental results shown in B, EGFR band intensities of far-Western blot (X-axis) and average Ct values of SH2-PLA (Y-axis) in panel B were plotted and showed strong correlation. a.u., arbitrary unit; r, Pearson correlation coefficient. d, Application of SH2-PLA for cancer tissue analysis. The SH2-PLA/Western/far-Western analyses were performed using 10 lung cancer tissue samples. Upper panel shows Western and far-Western results with antibody/probe names indicated on the left. Only one sample (#3) shows an EGFR size band which also overlapped with bands detected by anti-pTyr and Grb2 SH2 (far-Western image represents 60-min exposure). The tyrosine phosphorylation level of the band is similar to the weak phosphorylation of EGFR in unstimulated A431 cells (right panel). The PLA-SH2 results for the same set of lung cancer samples are shown on the bottom. Consistent with the Grb2 far-Western result, only sample #3 had significant signal beyond the no protein control (NPC). The BG line indicates the background Ct value

Mentions: Since the lysate dilution experiment indicated that the lysate requirement for SH2-PLA is very low, we next determined the lower limit of the assay by cell numbers. Serially diluted A431 cells were seeded in a 96-well plate, starved 16 h, and stimulated with EGF. Cells were lysed in the same volume of buffer and interaction between EGFR and Vav2 SH2 was analyzed by SH2-PLA. As shown in Fig. 4a, the assay detected EGF dependent SH2 interaction in the lysate equivalent of 16 cells (780 cells per well lysed in 50 μl) or approximately 2.5 ng which is close to the detection limit calculated above (Fig. 2c). In addition to EGFR-overexpressing A431 cells, we performed a similar lysate dilution experiment using EGF stimulated Cos1 cells and found that a lysate concentration approximately four times higher is required for detection, consistent with modest EGFR phosphorylation in Cos1 cells (Additional file 1: Figure S4 and data not shown). These results demonstrate that SH2-PLA is capable of detecting interaction between SH2 domains and pEGFR using a very small number of cultured cells.Fig. 4


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)

Applications of SH2-PLA assay. a, Practical limit of detection from cell culture. Two-fold serial dilutions of A431 cells were seeded to wells in a 96-well plate. Image series shows various 10x magnifications of diluted A431 cells with cell number indicated. Vav2 SH2:pEGFR interaction of starved or EGF-stimulated cell lysates was quantified by the SH2-PLA assay to resolve the assay detection limit. The Ct values from real-time PCR are shown with approximate numbers of cells per culture well or per assay (in brackets) underneath the chart. b, Time course and dose response of EGF stimulation. A431 and Cos1 cells were starved and stimulated with EGF at various times and concentrations as indicated. Upper panel shows far-Western blotting with Grb2 SH2 (25 μg lysate loaded per lane) and control blotting with anti-actin. Bottom panel shows Ct values of comparable SH2-PLA experiments loading 0.4 μg lysate per assay well. c, Correlation between far-Western and SH2-PLA assay. Using experimental results shown in B, EGFR band intensities of far-Western blot (X-axis) and average Ct values of SH2-PLA (Y-axis) in panel B were plotted and showed strong correlation. a.u., arbitrary unit; r, Pearson correlation coefficient. d, Application of SH2-PLA for cancer tissue analysis. The SH2-PLA/Western/far-Western analyses were performed using 10 lung cancer tissue samples. Upper panel shows Western and far-Western results with antibody/probe names indicated on the left. Only one sample (#3) shows an EGFR size band which also overlapped with bands detected by anti-pTyr and Grb2 SH2 (far-Western image represents 60-min exposure). The tyrosine phosphorylation level of the band is similar to the weak phosphorylation of EGFR in unstimulated A431 cells (right panel). The PLA-SH2 results for the same set of lung cancer samples are shown on the bottom. Consistent with the Grb2 far-Western result, only sample #3 had significant signal beyond the no protein control (NPC). The BG line indicates the background Ct value
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4482279&req=5

Fig4: Applications of SH2-PLA assay. a, Practical limit of detection from cell culture. Two-fold serial dilutions of A431 cells were seeded to wells in a 96-well plate. Image series shows various 10x magnifications of diluted A431 cells with cell number indicated. Vav2 SH2:pEGFR interaction of starved or EGF-stimulated cell lysates was quantified by the SH2-PLA assay to resolve the assay detection limit. The Ct values from real-time PCR are shown with approximate numbers of cells per culture well or per assay (in brackets) underneath the chart. b, Time course and dose response of EGF stimulation. A431 and Cos1 cells were starved and stimulated with EGF at various times and concentrations as indicated. Upper panel shows far-Western blotting with Grb2 SH2 (25 μg lysate loaded per lane) and control blotting with anti-actin. Bottom panel shows Ct values of comparable SH2-PLA experiments loading 0.4 μg lysate per assay well. c, Correlation between far-Western and SH2-PLA assay. Using experimental results shown in B, EGFR band intensities of far-Western blot (X-axis) and average Ct values of SH2-PLA (Y-axis) in panel B were plotted and showed strong correlation. a.u., arbitrary unit; r, Pearson correlation coefficient. d, Application of SH2-PLA for cancer tissue analysis. The SH2-PLA/Western/far-Western analyses were performed using 10 lung cancer tissue samples. Upper panel shows Western and far-Western results with antibody/probe names indicated on the left. Only one sample (#3) shows an EGFR size band which also overlapped with bands detected by anti-pTyr and Grb2 SH2 (far-Western image represents 60-min exposure). The tyrosine phosphorylation level of the band is similar to the weak phosphorylation of EGFR in unstimulated A431 cells (right panel). The PLA-SH2 results for the same set of lung cancer samples are shown on the bottom. Consistent with the Grb2 far-Western result, only sample #3 had significant signal beyond the no protein control (NPC). The BG line indicates the background Ct value
Mentions: Since the lysate dilution experiment indicated that the lysate requirement for SH2-PLA is very low, we next determined the lower limit of the assay by cell numbers. Serially diluted A431 cells were seeded in a 96-well plate, starved 16 h, and stimulated with EGF. Cells were lysed in the same volume of buffer and interaction between EGFR and Vav2 SH2 was analyzed by SH2-PLA. As shown in Fig. 4a, the assay detected EGF dependent SH2 interaction in the lysate equivalent of 16 cells (780 cells per well lysed in 50 μl) or approximately 2.5 ng which is close to the detection limit calculated above (Fig. 2c). In addition to EGFR-overexpressing A431 cells, we performed a similar lysate dilution experiment using EGF stimulated Cos1 cells and found that a lysate concentration approximately four times higher is required for detection, consistent with modest EGFR phosphorylation in Cos1 cells (Additional file 1: Figure S4 and data not shown). These results demonstrate that SH2-PLA is capable of detecting interaction between SH2 domains and pEGFR using a very small number of cultured cells.Fig. 4

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