Analysis of Individual Signaling Complexes by mMAPS, a Flow‐Proteometric System

Chao‐Kai Chou1, Pei‐Hsiang Tsou1, Jennifer L. Hsu2, Heng‐Huan Lee3, Ying‐Nai Wang2, Jun Kameoka4, Mien‐Chie Hung2

1 These authors contributed equally to this work, 2 Department of Biotechnology, Asia University, Taichung, Taiwan, 3 Department of Molecular and Cellular Oncology, University of Texas MD Anderson Cancer Center, Houston, Texas, 4 Department of Electrical and Computer Engineering, Texas A&M University, College Station, Texas
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 20.11
DOI:  10.1002/0471142727.mb2011s114
Online Posting Date:  April, 2016
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Signal transduction is essential for maintaining normal cell physiological functions, and deregulation of signaling can lead to diseases such as diabetes and cancers. Some of the major players in signal delivery are molecular complexes composed of proteins and nucleic acids. This unit describes a technique called microchannel for multiparameter analysis of proteins in a single complex (mMAPS) for analyzing and quantifying individual target signaling complexes. mMAPS is a flow‐proteometric system that allows detection of individual proteins or complexes flowing through a microfluidic channel. Specific target proteins and nucleic acids labeled by fluorescent tags are harvested from tissues or cultured cells for analysis by the mMAPS system. Overall, mMAPS enables both detection of multiple components within a single complex and direct quantification of different populations of molecular complexes in one setting in a short timeframe and requiring very low sample input. © 2016 by John Wiley & Sons, Inc.

Keywords: single molecule; single complex; protein‐protein interaction; protein−nucleic acid interaction; flow proteometry; microfluidic

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Setting Up mMAPS Analysis for Single Complex Measurements in Cultured Cells
  • Alternate Protocol 1: Preparation of Tissue Lysates for mMAPS Analysis
  • Support Protocol 1: Fabrication of Microfluidic Channel Devices
  • Support Protocol 2: Optical Calibration of the Instrument
  • Basic Protocol 2: Analysis of mMAPS Data
  • Reagents and Solutions
  • Commentary
  • Figures
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Basic Protocol 1: Setting Up mMAPS Analysis for Single Complex Measurements in Cultured Cells

  • Cells of interest
  • Culture medium
  • Phosphate‐buffered saline (PBS; appendix 22)
  • 1% (v/v) paraformaldehyde (PFA; Electron Microscopy Sciences, cat. no. 15710) in PBS
  • 125 mM glycine (Fisher, cat. no. BP3815) in PBS
  • 0.25% (v/v) Triton X‐100 (Sigma, cat. no. T8787) in PBS
  • 6% (w/v) bovine serum albumin (BSA; Gemini BioProducts, cat. no. 700100P1KG) in PBS
  • Fluorescently conjugated primary antibody or unlabeled primary antibody with fluorescently labeled secondary antibody
  • Nucleic acid dye (e.g., TOTO3; Life Technologies) or other dye of interest
  • Lysis buffer (see recipe)
  • Detection buffer (see recipe)
  • Coating buffer (see recipe)
  • 24‐well tissue culture microplate
  • Orbital shaker
  • Cell scraper
  • 1.5‐ml microcentrifuge tubes
  • Sonicator (e.g., Diagenode, Bioruptor)
  • NanoDrop 2000 spectrophotometer (Thermo Scientific) or equivalent
  • Highly sensitive photon‐detecting instrument (e.g., Alba Confocal Spectroscopy and Imaging Station, ISS)
  • UV‐grade quartz microfluidic channel device, 2‐μm wide (cross‐section) by 0.5 μm deep at detection points (see protocol 2)
  • High voltage power supply (Stanford Research Systems, cat. no. PS350/5000 V‐25 W)
  • Gold electrodes (Scientific Instrument Services, cat. no. W352)
CAUTION: Paraformaldehyde is toxic if inhaled or swallowed; is irritating to the skin, eyes, and respiratory system; and may be carcinogenic. Paraformaldehyde should be used with appropriate safety measures such as protective gloves, glasses, clothing, and sufficient ventilation. Waste should be handled according to local hazardous waste regulations. Detailed information can be found in MSDS.

Alternate Protocol 1: Preparation of Tissue Lysates for mMAPS Analysis

  Additional Materials (also see protocol 1)
  • Tissue of interest (e.g., tumor, ≥8 mm in diameter)
  • Optimum cutting temperature (OCT) compound (Tissue‐Tek, cat. no. 4583)
  • Freezing microtome
  • Glass microscope slides
  • Mini PAP pen (Invitrogen, cat. no. 008877)
  • Slide chamber

Support Protocol 1: Fabrication of Microfluidic Channel Devices

  • Acetone
  • Isopropanol
  • Pressurized nitrogen
  • Microposit S1813 positive photoresist
  • Microposit MF‐319 developer
  • Piranha solution: 3:1 (v/v) H 2SO 4/H 2O 2 (J.T. Baker, cat. nos. 9681 and 2186)
  • 170‐ and 500‐μm‐thick ultraviolet (UV)‐grade quartz wafers (both 4 inches in diameter; Mark Optics)
  • Tweezers
  • Spin coater (Laurell, WS‐650 S Spin Processor)
  • Laboratory hot plate
  • Furnace that can reach 1050°C (Cole‐Parmer, EW‐33855‐30)
  • Photomask with custom‐designed microchannel patterns (Cornell NanoScale Science & Technology Facility [CNF])
  • Mask aligner (Karl Suss, MA6 Mask Aligner)
  • Reactive ion etching (RIE) system (Oxford, Plasmalab 100 ICP RIE System)
  • Drilling press (e.g., Ryobi)
  • Reservoirs (Western Analytical, NanoPort Reservoir Assemblies N‐131)

Support Protocol 2: Optical Calibration of the Instrument

  • 10 nM individual solutions of Alexa Fluor 488, Alexa Fluor 555, and Alexa Fluor 647 (Life Technologies, A11029, A21434, and A21236) in PBS ( appendix 22)
  • Nunc Lab‐Tek Chambered Coverglass, 8‐well (Thermo Scientific, 12565470)
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Literature Cited

Literature Cited
  Auerbach, D., Thaminy, S., Hottiger, M.O., and Stagljar, I. 2002. The post‐genomic era of interactive proteomics: Facts and perspectives. Proteomics 2:611‐623. doi: 10.1002/1615‐9861(200206)2:6%3c611::AID‐PROT611%3e3.0.CO;2‐Y.
  Campbell, S.A. 2007. The Science and Engineering of Microelectronic Fabrication, 2nd ed. Oxford University Press, New Delhi.
  Cheng, Y., Ho, E., Subramanyam, B., and Tseng, J.L. 2004. Measurements of drug‐protein binding by using immobilized human serum albumin liquid chromatography‐mass spectrometry. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 809:67‐73. doi: 10.1016/j.jchromb.2004.06.006.
  Chou, C.K., Jing, N., Yamaguchi, H., Tsou, P.H., Lee, H.H., Chen, C.T., Wang, Y.N., Hong, S., Su, C., Kameoka, J., and Hung, M.C. 2010. High speed digital protein interaction analysis using microfluidic single molecule detection system. Lab. Chip 10:1793‐1798. doi: 10.1039/c002937h.
  Chou, C.K., Lee, H.H., Tsou, P.H., Chen, C.T., Hsu, J.M., Yamaguchi, H., Wang, Y.N., Lee, H.J., Hsu, J.L., Lee, J.F., Kameoka, J., and Hung, M.C. 2014. mMAPS: A flow‐proteometric technique to analyze protein‐protein interactions in individual signaling complexes. Sci. Signal. 7:rs1. doi: 10.1126/scisignal.2004620.
  Cipriany, B.R., Zhao, R., Murphy, P.J., Levy, S.L., Tan, C.P., Craighead, H.G., and Soloway, P.D. 2010. Single molecule epigenetic analysis in a nanofluidic channel. Anal. Chem. 82:2480‐2487. doi: 10.1021/ac9028642.
  Collas, P. 2009. The state‐of‐the‐art of chromatin immunoprecipitation. Methods Mol. Biol. 567:1‐25. doi: 10.1007/978‐1‐60327‐414‐2_1.
  Colyer, R.A., Scalia, G., Kim, T., Rech, I., Resnati, D., Marangoni, S., Ghioni, M., Cova, S., Weiss, S., and Michalet, X. 2010. High‐throughput multispot single‐molecule spectroscopy. Proc. Soc. Photo Opt. Instrum. Eng. 7571:75710G‐75710G75711.
  Doherty, E.A., Meagher, R.J., Albarghouthi, M.N., and Barron, A.E. 2003. Microchannel wall coatings for protein separations by capillary and chip electrophoresis. Electrophoresis 24:34‐54. doi: 10.1002/elps.200390029.
  Gavin, A.C. and Superti‐Furga, G. 2003. Protein complexes and proteome organization from yeast to man. Curr. Opin. Chem. Biol. 7:21‐27. doi: 10.1016/S1367‐5931(02)00007‐8.
  Haring, M., Offermann, S., Danker, T., Horst, I., Peterhansel, C., and Stam, M. 2007. Chromatin immunoprecipitation: Optimization, quantitative analysis and data normalization. Plant Methods 3:11. doi: 10.1186/1746‐4811‐3‐11.
  Hogan, G.J., Lee, C.K., and Lieb, J.D. 2006. Cell cycle‐specified fluctuation of nucleosome occupancy at gene promoters. PLoS Genet. 2:e158. doi: 10.1371/journal.pgen.0020158.
  Jain, A., Liu, R., Ramani, B., Arauz, E., Ishitsuka, Y., Ragunathan, K., Park, J., Chen, J., Xiang, Y.K., and Ha, T. 2011. Probing cellular protein complexes using single‐molecule pull‐down. Nature 473:484‐488. doi: 10.1038/nature10016.
  Kameoka, J., Craighead, H.G., Zhang, H.W., and Henion, J. 2001. A polymeric microfluidic chip for CE/MS determination of small molecules. Anal. Chem. 73:1935‐1941. doi: 10.1021/ac001533t.
  Kolch, W. and Pitt, A. 2010. Functional proteomics to dissect tyrosine kinase signalling pathways in cancer. Nat. Rev. Cancer 10:618‐629. doi: 10.1038/nrc2900.
  Kuo, M.H. and Allis, C.D. 1999. In vivo cross‐linking and immunoprecipitation for studying dynamic protein:DNA associations in a chromatin environment. Methods 19:425‐433. doi: 10.1006/meth.1999.0879.
  Lee, N.K., Kapanidis, A.N., Koh, H.R., Korlann, Y., Ho, S.O., Kim, Y., Gassman, N., Kim, S.K., and Weiss, S. 2007. Three‐color alternating‐laser excitation of single molecules: Monitoring multiple interactions and distances. Biophys. J. 92:303‐312. doi: 10.1529/biophysj.106.093211.
  Liu, C., Qu, Y., Luo, Y., and Fang, N. 2011. Recent advances in single‐molecule detection on micro‐ and nano‐fluidic devices. Electrophoresis 32:3308‐3318. doi: 10.1002/elps.201100159.
  Michalet, X., Colyer, R.A., Scalia, G., Kim, T., Levi, M., Aharoni, D., Cheng, A., Guerrieri, F., Arisaka, K., Millaud, J., Rech, I., Resnati, D., Marangoni, S., Gulinatti, A., Ghioni, M., Tisa, S., Zappa, F., Cova, S., and Weiss, S. 2010. High‐throughput single‐molecule fluorescence spectroscopy using parallel detection. Proc. Soc. Photo Opt. Instrum. Eng. 7608. doi: 10.1117/12.846784.
  Myers, J.D. 1989. Development and application of immunocytochemical staining techniques: A review. Diagn. Cytopathol. 5:318‐330. doi: 10.1002/dc.2840050317.
  Nolan, R.L., Cai, H., Nolan, J.P., and Goodwin, P.M. 2003. A simple quenching method for fluorescence background reduction and its application to the direct, quantitative detection of specific mRNA. Anal. Chem. 75:6236‐6243. doi: 10.1021/ac034803r.
  Papin, J.A., Hunter, T., Palsson, B.O., and Subramaniam, S. 2005. Reconstruction of cellular signalling networks and analysis of their properties. Nat. Rev. Mol. Cell Biol. 6:99‐111. doi: 10.1038/nrm1570.
  Phizicky, E.M. and Fields, S. 1995. Protein‐protein interactions: Methods for detection and analysis. Microbiol. Rev. 59:94‐123.
  Stenman, U.H., Bidart, J.M., Birken, S., Mann, K., Nisula, B., and O'Connor, J. 1993. Standardization of protein immunoprocedures. Choriogonadotropin (CG). Scand. J. Clin. Lab. Invest. Suppl. 216:42‐78. doi: 10.3109/00365519309086908.
  Thompson, M.A., Lew, M.D., and Moerner, W.E. 2012. Extending microscopic resolution with single‐molecule imaging and active control. Annu. Rev. Biophys. 41:321‐342. doi: 10.1146/annurev‐biophys‐050511‐102250.
  Toby, G.G. and Golemis, E.A. 2001. Targeting proteins to specific cellular compartments to optimize physiological activity. Methods Enzymol. 332:77‐87. doi: 10.1016/S0076‐6879(01)32193‐6.
  Williams, N.E. 2000. Immunoprecipitation procedures. Methods Cell Biol. 62:449‐453. doi: 10.1016/S0091‐679X(08)61549‐6.
Key Reference
  Chou et al., 2014. See above.
  Describes the first application of mMAPS in analyzing individual signaling complexes from tissue and cell culture samples.
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