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mMAPS: A Flow-Proteometric Technique to Analyze Protein-Protein Interactions in Individual Signaling Complexes

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Science Signaling  04 Mar 2014:
Vol. 7, Issue 315, pp. rs1
DOI: 10.1126/scisignal.2004620
  • Fig. 1 Schematic diagram of mMAPS single signaling complex detection.

    (A) After labeling fluorophores to target molecules, sample lysate was loaded into the microchannel and flowed through the detection spot in the center of the microchannel. Target molecules or complexes were excited by three lasers with wavelengths of 405, 488, and 637 nm. Emission fluorescence was observed by three APDs, which specifically detect three different spectral ranges (531/40 nm for GFP and A488, 685/40 nm for A647 and TOTO3, and 605/15 nm for QD605). The photon burst signals were then analyzed to determine the complex formation. (B) Diagram of photon bursts of the detected complex. Left: All three fluorophores were detected simultaneously, indicating that three target molecules were within a complex. Middle: Two of the three fluorophores showed coincidently signals, indicating a two-target molecule interaction. Right: No coincident signal represents individual target molecule alone.

  • Fig. 2 Direct lysate analysis of EGF-EGFR complex in cells overexpressing EGFR.

    After HeLa cells overexpressing EGFRGFP were treated with EGFA647 (100 ng/ml) for 30 min, the lysate was harvested and directly analyzed by mMAPS. (A) A 15-s photon burst profile showing EGFRGFP and EGFA647 signals. (B) Representative zoom-in views of typical events over a 0.5-s time frame. Top: EGFRGFP not bound to EGFA647. Bottom: Coincidence of EGFRGFP and EGFA647 signals, indicating the complex. (C and D) Representative 2D plot showing the fluorescence of detected events during a 20-min flow-proteometric analysis without (C) or with (D) EGF treatment. (E) Ratio of EGFA647-bound EGFRGFP (EGFRA647-EGFRGFP interaction eventstotal EGFRGFP interaction events×100; “EGFA647-EGFRGFP” means EGFA647 and EGFRGFP interaction complex; total EGFRGFP events included unbound and EGFA647-bound EGFRGFP events) to all detected EGFR in the presence and absence of EGF. Data are means ± SEM of three independent experiments (others shown in fig. S3).

  • Fig. 3 Detection and quantification of individual p53GFP and genomic DNA interaction.

    (A) Representative photon bursts of a p53GFP-alone event (left) and an event of p53GFP interacting with genomic DNA (right) in a lysate from HeLa cells expressing p53GFP and stained with the DNA dye TOTO3. (B) Representative 2D plot showing the fluorescence distribution and the quantities of p53GFP, genomic DNA fragments, and the interaction complexes. Each plot represents a 20-min flow-proteometric analysis, and the detection was repeated three times. The numbers in parentheses are the quantities of specific events. (C) Proportion of genomic DNA-bound and unbound p53GFP among all detected p53GFP (p53GFP-DNA interaction eventstotal p53GFP events×100). Data are means ± SEM of three independent experiments.

  • Fig. 4 Detection and quantification of individual p53GFP with Ser15 phosphorylation or Lys372 methylation.

    (A) Representative 2D plot showing the fluorescence distribution and the quantities of p53GFP and p53GFP-pSer15 events in HeLa cells expressing p53GFP. Each plot represents a 20-min flow-proteometric detection, and the numbers in parentheses are the quantities of specific events. (B) Ratio of p53GFP labeled with the pSer15 antibody to all detected p53GFP events (p53GFPpSer15 eventstotal p53GFP events×100). (C) Representative 2D plot showing the fluorescence distribution and the quantities of p53GFP and p53GFPmeLys372 events. (D) Ratio of p53GFPmeLys372 among all detected p53GFP (p53GFPmeLys372 eventstotal p53GFP events×100). (E) Representative 2D plot of p53GFP and control IgG events. (F) Ratio of control IgG-labeled p53GFP. Data are means ± SEM of three independent experiments.

  • Fig. 5 Analysis of the interaction between endogenous EGFR and EGFA647.

    (A) 2D plots showing the detection of endogenous phosphorylated (pTyr1068) and nonphosphorylated EGFR in complex with EGF in serum-starved HeLa cells (0 min) and 30 to 120 min after addition of EGFA647. The numbers in parentheses are the quantities of the indicated target-specific events. (B) Ratio of EGFR-pTyr1068 (EGFR-pTyr1068 eventstotal EGFR events×100; yellow), EGF-EGFR (EGF-EGFR” interaction eventstotal EGFR events×100; blue), and EGF–EGFR-pTyr1068 EGF–EGFR-pTyr1068 interaction eventstotal EGFR events×100; black), among all detected EGFR events from (A). EGF stimulated for 0 min (left), 30 min (middle), and 120 min (right). Data are means ± SEM of three independent experiments.

  • Fig. 6 mMAPS analysis of endogenous EGFR, STAT3, and associated complexes in tumor tissue sample.

    (A) 2D plot showing the fluorescence distribution and the quantities of EGFR, STAT3, and EGFR-STAT3 in tumor tissue during a 20-min flow-proteometric analysis. (B) 2D plot of nonspecific binding control by using normal rabbit and goat IgG as the primary antibodies to stain consecutive tissue section samples. (C) Mean ratios ± SEM of the EGFR-STAT3 interaction to total detected STAT3 (EGFR-STAT3” interaction eventstotal STAT3 events×100) or EGFR (EGFR-STAT3” interaction eventstotal EGFR events×100). Data are representative or means ± SEM of three independent experiments.

  • Fig. 7 Analysis of genomic DNA, endogenous STAT3, p300, and associated complexes in tumor.

    (A) 3D plot of STAT3, p300, DNA, and associated complexes with their quantities and fluorescence distribution during a 20-min flow-proteometric analysis. The numbers in parentheses are the numbers of events. (B) Control samples were stained with normal goat and rabbit IgGs without TOTO3 stain. (C) Left: Mean ratios ± SEM of STAT3-associated complexes with DNA (STAT3-DNA” interaction eventstotal STAT3 events×100), with p300 (STAT3-p300” interaction eventstotal STAT3 events×100), or with both (STAT3-p300-DNA” interaction eventstotal STAT3 events×100) to total detected STAT3 molecules. Right: Mean ratios ± SEM of p300-associated complexes with STAT3 (STAT3-p300” interaction eventstotal p300 events×100), with DNA (p300-DNA” interaction eventstotal p300 events×100), or both (STAT3-p300-DNA” interaction eventstotal p300 events×100) to total detected p300 molecules. Data are representative or means ± SEM of three independent experiments.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/7/315/rs1/DC1

    Fig. S1. Workflow for directly detecting exogenous protein complexes in lysates.

    Fig. S2. 2D plots of the repeated mMAPS analysis of EGFA647 and EGFRGFP interaction.

    Fig. S3. Photon burst profile of genomic DNA harvested from cell lysate.

    Fig. S4. 2D plots of the repeated mMAPS analysis of individual p53GFP and genomic DNA interaction.

    Fig. S5. 2D plots of the repeated mMAPS analysis of individual p53GFP with either Ser15 phosphorylation, Lys372 methylation, or the control IgG.

    Fig. S6. Workflow for directly detecting immobilized endogenous proteins in lysates or tissue.

    Fig. S7. mMAPS analysis for testing the recognition of EGFRGFP by the D38 EGFR antibody.

    Fig. S8. mMAPS analysis for testing the recognition of EGFRGFP by the ab13 EGFR antibody.

    Fig. S9. mMAPS analysis for testing the recognition of EGFRGFP by a Flag antibody as a negative control.

    Fig. S10. mMAPS analysis for testing the recognition of STAT3GFP by a STAT3 antibody.

    Fig. S11. 2D plots of the repeated mMAPS analysis of endogenous EGFR and EGFA647 interaction with or without EGFR-pTyr1068.

    Fig. S12. Images and repeats of mMAPS analysis of endogenous EGFR and STAT3 interactions in MDA-MB-468 mouse tumor xenograft tissue.

    Fig. S13. EGFR and STAT3 interaction ratio from tumor tissues of MDA-MB-468 breast cancer xenograft after antibody efficiency normalization.

    Fig. S14. Images and 3D plots of the repeated mMAPS analysis of endogenous STAT3, p300, and genomic DNA interactions in MDA-MB-468 mouse tumor xenograft tissue.

    Fig. S15. Photon burst profiles of specific fluorophores detected by mMAPS platform.

    Fig. S16. Determination of antibody labeling efficiency for mMAPS.

    Table S1. Data from each independent experiment used to quantify the ratios of complex formation presented in the figures.

  • Supplementary Materials for:

    mMAPS: A Flow-Proteometric Technique to Analyze Protein-Protein Interactions in Individual Signaling Complexes

    Chao-Kai Chou, Heng-Huan Lee, Pei-Hsiang Tsou, Chun-Te Chen, Jung-Mao Hsu, Hirohito Yamaguchi, Ying-Nai Wang, Hong-Jen Lee, Jennifer L. Hsu, Jin-Fong Lee, Jun Kameoka,* Mien-Chie Hung*

    *Corresponding author. E-mail: mhung@mdanderson.org (M.-C.H.); kameoka@mail.ece.tamu.edu (J.K.)

    This PDF file includes:

    • Fig. S1. Workflow for directly detecting exogenous protein complexes in lysates.
    • Fig. S2. 2D plots of the repeated mMAPS analysis of EGFA647 and EGFRGFP interaction.
    • Fig. S3. Photon burst profile of genomic DNA harvested from cell lysate.
    • Fig. S4. 2D plots of the repeated mMAPS analysis of individual p53GFP and genomic DNA interaction.
    • Fig. S5. 2D plots of the repeated mMAPS analysis of individual p53GFP with either Ser15 phosphorylation, Lys372 methylation, or the control IgG.
    • Fig. S6. Workflow for directly detecting immobilized endogenous proteins in lysates or tissue.
    • Fig. S7. mMAPS analysis for testing the recognition of EGFRGFP by the D38 EGFR antibody.
    • Fig. S8. mMAPS analysis for testing the recognition of EGFRGFP by the ab13 EGFR antibody.
    • Fig. S9. mMAPS analysis for testing the recognition of EGFRGFP by a Flag antibody as a negative control.
    • Fig. S10. mMAPS analysis for testing the recognition of STAT3GFP by a STAT3 antibody.
    • Fig. S11. 2D plots of the repeated mMAPS analysis of endogenous EGFR and EGFA647 interaction with or without EGFR-pTyr1068.
    • Fig. S12. Images and repeats of mMAPS analysis of endogenous EGFR and STAT3 interactions in MDA-MB-468 mouse tumor xenograft tissue.
    • Fig. S13. EGFR and STAT3 interaction ratio from tumor tissues of MDA-MB-468 breast cancer xenograft after antibody efficiency normalization.
    • Fig. S14. Images and 3D plots of the repeated mMAPS analysis of endogenous STAT3, p300, and genomic DNA interactions in MDA-MB-468 mouse tumor xenograft tissue.
    • Fig. S15. Photon burst profiles of specific fluorophores detected by mMAPS platform.
    • Fig. S16. Determination of antibody labeling efficiency for mMAPS.
    • Table S1. Data from each independent experiment used to quantify the ratios of complex formation presented in the figures.

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    Citation: C.-K. Chou, H.-H. Lee, P.-H. Tsou, C.-T. Chen, J.-M. Hsu, H. Yamaguchi, Y.-N. Wang, H.-J. Lee, J. L. Hsu, J.-F. Lee, J. Kameoka, M.-C. Hung, mMAPS: A Flow-Proteometric Technique to Analyze Protein-Protein Interactions in Individual Signaling Complexes. Sci. Signal. 7, rs1 (2014).

    © 2014 American Association for the Advancement of Science

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