Protein Microarrays for Identification of Novel Extracellular Protein‐Protein Interactions

Irene Tom1, Nicholas Lewin‐Koh2, Sree R. Ramani1, Lino C. Gonzalez1

1 Department of Protein Chemistry, Genentech, South San Francisco, California, 2 Department of Nonclinical Biostatistics, Genentech, South San Francisco, California
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 27.3
DOI:  10.1002/0471140864.ps2703s72
Online Posting Date:  April, 2013
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Abstract

Functional protein microarrays offer the capability for high‐throughput protein interaction analysis and have long promised to be a powerful tool for understanding protein interactions at the proteome scale. Although popular techniques for protein‐protein interaction mapping like yeast‐two‐hybrid and affinity‐purification mass spectrometry have performed well for identifying intracellular protein‐protein interactions, the study of interactions between extracellular proteins has remained challenging for these methods. Instead, the use of protein microarrays appears to be a robust and efficient method for the identification of interactions among the members of this class of protein. This unit describes methods for extracellular protein microarray production, screening, and analysis. A protocol is described for enhanced detection of low‐affinity interactions by generating multivalent complexes using Fc‐fusion bait proteins and protein A microbeads, along with a statistical method for hit scoring and identification of nonspecific interactions. Curr. Protoc. Protein Sci. 72:27.3.1‐27.3.24. © 2013 by John Wiley & Sons, Inc.

Keywords: protein microarray; microarray analysis; extracellular protein; multivalent binding; protein‐protein interaction

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Production of Extracellular Protein Microarrays
  • Basic Protocol 2: Preparation of Labeled Bait Protein and Multivalent Microbead Complexes
  • Alternate Protocol 1: Formation of Cy5‐Labeled IgG and Fc‐Fusion Bait Protein Multivalent Complexes on Protein A Microbeads
  • Basic Protocol 3: Protein Microarray Screening and Processing
  • Basic Protocol 4: Protein Microarray Data Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Production of Extracellular Protein Microarrays

  Materials
  • Purified proteins
  • PBS, pH 7.2 (see recipe)
  • 80% (v/v) glycerol (Ultrapure MB grade; USB corporation, cat. no. 56‐81‐5) in PBS (see recipe for PBS)
  • 40% and 50% (v/v) glycerol in PBS
  • 20% (v/v) ethanol
  • ZeptoMARK blocking buffer BB1 (Zeptosens, cat. no. 9040)
  • Bovine serum albumin fraction V, heat shock, fatty‐acid free (Roche Applied Science, cat. no. 03‐117‐057‐001; optional)
  • 5% milk in PBST (see recipe)
  • PBST (see recipe)
  • Low‐protein‐binding 0.2‐µm membrane filters (PALL Life Sciences)
  • Polypropylene cryogenic vials, 1.2 ml (Corning, cat. no. 430658)
  • Polypropylene, conical 96‐well plates (Greiner Bio‐One, cat. no. 82050‐678)
  • Adhesive aluminum foil seal (AlumaSeal 384; Excel Scientific, cat. no. F‐384‐100)
  • 8‐channel pipettor (20‐µl capacity; various vendors)
  • Polypropylene, conical 384‐well plates (Arrayit, cat. no. MMP384)
  • NanoPrint LM60 (Arrayit) or a similar 48‐pin contact microarrayer
  • Micro Spotting Pins (Arrayit, cat. no. 946MP3)
  • Epoxysilane‐coated glass slides (Nexterion Slide E; Schott, cat. no. 1064016)
  • Gloves (powder‐free chloroprene; Microflex, cat. no. NEC‐288)
  • ZeptoFOG blocking station (Zeptosens, cat. no. 1210)
  • Glass holder and slide rack set (Wheaton, cat. no. 900303)
  • Additional reagents and equipment for preparing labeled protein ( protocol 2)

Basic Protocol 2: Preparation of Labeled Bait Protein and Multivalent Microbead Complexes

  Materials
  • Cy5 monoreactive dye pack (GE Healthcare, cat. no. PA25001)
  • PBS (see recipe)
  • Bait protein: can be any extracellular protein of interest to the investigator, e.g., an orphan receptor that functions in cellular communication and regulation but whose ligand(s) are unknown
  • 5% milk in PBST (see recipe)
  • Protein A MicroBeads (Miltenyi Biotec, cat. no. 120‐000‐396)
  • End‐over‐end rotator
  • Pro‐Spin columns (Princeton Separations, cat. no. CS‐800)
  • Tabletop ultracentrifuge (Beckman Coulter, model TL‐100)
  • Nanodrop spectrophotometer (Thermo Fisher Scientific; also see unit 3.10)
  • Octet protein A sensors (ForteBio, cat. no. 18‐5010; http://www.fortebio.com/)
  • Octet biolayer inferometer (ForteBio; http://www.fortebio.com/)
  • 96‐well black polypropylene plates (Greiner Bio‐One, cat. no. 655209)

Alternate Protocol 1: Formation of Cy5‐Labeled IgG and Fc‐Fusion Bait Protein Multivalent Complexes on Protein A Microbeads

  • Human IgG (Jackson ImmunoResearch, cat. no. 009‐000‐003)

Basic Protocol 3: Protein Microarray Screening and Processing

  Materials
  • Printed protein microarray slides ( protocol 1)
  • PBST (see recipe)
  • Protein A (Sigma‐Aldrich, cat. no. P7837)
  • 5% milk in PBST (see recipe)
  • PBS (see recipe)
  • Forceps
  • Hybridization station (Miltenyi Biotec, a‐Hyb or similar)
  • 50‐ml conical tubes (e.g., BD Falcon)
  • Swinging‐bucket centrifuge
  • GenePix 4000B microarray scanner (Molecular Devices) or equivalent
  • GenePix Pro 6.0 software (Molecular Devices) or equivalent data extraction software

Basic Protocol 4: Protein Microarray Data Analysis

  Materials
  • Mac/PC or Unix/Linux server running R
  • Ellipse, Limma and gpscreen R packages (links provided below)
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Figures

Videos

Literature Cited

Literature Cited
   Bushell, K.M., Sollner, C., Schuster‐Boeckler, B., Bateman, A., and Wright, G.J. 2008. Large‐scale screening for novel low‐affinity extracellular protein interactions. Genome Res. 18:622‐630.
   Clark, H.F., Gurney, A.L., Abaya, E., Baker, K., Baldwin, D., Brush, J., Chen, J., Chow, B., Chui, C., Crowley, C., Currell, B., Deuel, B., Dowd, P., Eaton, D., Foster, J., Grimaldi, C., Gu, Q., Hass, P.E., Heldens, S., Huang, A., Kim, H.S., Klimowski, L., Jin, Y., Johnson, S., Lee, J., Lewis, L., Liao, D., Mark, M., Robbie, E., Sanchez, C., Schoenfeld, J., Seshagiri, S., Simmons, L., Singh, J., Smith, V., Stinson, J., Vagts, A., Vandlen, R., Wantanabe, C., Wiend, D., Woods, K., Xie, M.H., Yansura, D., Yi, S., Yu, G., Yuan, J., Zhang, M., Zhang, Z., Gaddard, A., Wood, W.I., Godowski, P., and Gray, A. 2003. The secreted protein discovery initiative (SPDI), a large‐scale effort to identify novel human secreted and transmembrane proteins: A bioinformatics assessment. Genome Res. 13:2265‐2270.
   Letarte, M., Voulgaraki, D., Hatherley, D., Foster‐Cuevas, M., Saunders, N.J., and Barclay, A.N. 2005. Analysis of leukocyte membrane protein interactions using protein microarrays. BMC Biochem. 6:2.
   Ramani, S.R., Tom, I., Lewin‐Koh, N., Wranik, B., DePalatis, L., Zhang, J., Eaton, D., and Gonzalez, L.C. 2012. A secreted protein microarray platform for extracellular protein interaction discovery. Anal. Biochem. 420:127‐138.
   Silver, J., Ritchie, M. E., and Smyth, G. K. 2009. Microarray background correction: Maximum likelihood estimation for the normal‐exponential convolution model. Biostatistics 10:352‐363.
   Smyth, G. K. 2005. Limma: Linear models for microarray data. In Bioinformatics and Computational Biology Solutions using R and Bioconductor (R. Gentleman, V. Carey, S. Dudoit, R. Irizarry, and W. Huber, eds.) pp. 397‐420. Springer, New York.
   Sun, Y., Gallagher‐Jones, M., Barker, C., and Wright, G.J. 2012. A benchmarked protein microarray‐based platform for the identification of novel low‐affinity extracellular protein interactions. Anal. Biochem. 424:45‐53.
   Voulgaraki, D., Mitnacht‐Kras, R., Letarte, M., Foster‐Cuevas, M., Brown, M.H., and Barclay, A.N. 2005. Multivalent recombinant proteins for probing functions of leukocyte surface proteins such as the CD200 receptor. Immunology 115:337‐346.
   Wright, G.J. 2009. Signal initiation I biological systems: the properties and detection of transient extracellular protein interactions. Mol. Biosyst. 5:1405‐1412.
   Wright, G.J., Martin, S., Bushell, K.M., and Sollner, C. 2010. High‐throughput identification of transient extracellular protein interactions. Biochem. Soc. Trans. 38:919‐922.
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