Post‐Translational Modification Profiling—a High‐Content Assay for Identifying Protein Modifications in Mammalian Cellular Systems

Yifat Merbl1, Marc W. Kirschner1

1 Department of Systems Biology, Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 27.8
DOI:  10.1002/0471140864.ps2708s77
Online Posting Date:  August, 2014
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Abstract

Protein microarrays are extremely useful for detecting substrates of phosphorylation, substrates of ubiquitylation, or other post‐translational modifications. The ability to screen binding interactions as well as post‐translational modifications of thousands of proteins at once has improved our ability to identify their targets. Utilizing such systems in combination with functional mammalian cell extracts that preserve enzymatic activity offers advantages in identifying semi‐quantitative changes of these interactions in the context of specific cellular conditions. This unit provides a detailed procedure for setting up an extract‐based activity assay for high content detection of protein post‐translation modifications. It also provides basic guidelines for data analysis. Curr. Protoc. Protein Sci. 77:27.8.1‐27.8.13 © 2014 by John Wiley & Sons, Inc.

Keywords: PTM profiling; post‐translational modification; functional assay; ubiquitin (Ub); protein microarray

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: PTM Profiling Using Mammalian Cell Extracts and Protein Microarrays
  • Support Protocol 1: Preparation of Functional Mammalian Cell Extracts
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: PTM Profiling Using Mammalian Cell Extracts and Protein Microarrays

  Materials
  • ProtoArray (Life Technologies): while protein microarrays are available from other suppliers, the protocol described herein is based on the use of this particular product.
  • TBS, cold (see recipe)
  • TBS‐T: TBS (see recipe) with 0.05% (v/v) Tween‐20
  • ArrayIt blocking buffer, cold (ArrayIt Corporation)
  • Functional extracts (see Support Protocol)
  • Recombinant proteins to supplement extracts: can be purified or bought commercially (Boston Biochem)
  • Primary antibody to detect PTM of interest (e.g., FK1 poly‐ubiquitin antibody; BioMol)
  • Fluorescently labeled secondary antibody (Jackson Immunoresearch Laboratories)
  • 24 × 60 mm cover slips
  • Forceps
  • Eppendorf centrifuge 5810 and rotor A‐4‐81 for 50‐ml conical tubes (or equivalent)
  • GenePix 4000B microarray scanner (Molecular Devices)
  • Gene Pix Pro software (Molecular Devices)
  • Optional: ProtoArray Prospector v.5 (available at http://www.lifetechnologies.com/us/en/home/life‐science/protein‐expression‐and‐analysis/biomarker‐discovery/protoarray/resources/data‐analysis.html)
NOTE: It is highly recommended to read the user's manual of the human ProtoArray (Life Technologies) before starting this protocol.

Support Protocol 1: Preparation of Functional Mammalian Cell Extracts

  Materials
  • HeLa S3 cells (ATCC) grown in suspension, or HeLa or other cell line grown on plates
  • Dulbecco's Modified Eagle's Medium (DMEM) (Corning, cat. no. 10‐013)
  • FBS (Life Technologies, cat. no. 26140‐079)
  • PBS for cell culture (Corning, cat. no. 21‐040, or equivalent)
  • Antibiotic/antimycotic solution (Corning, cat. no. 30‐004‐CI)
  • 0.25% Trypsin/1 mM EDTA (Life Technologies, cat. no. 25200‐056)
  • 200 mM thymidine: prepare fresh in sterile water and filter‐sterilize with 0.22 µm filter
  • 5 mg/ml nocodazole in DMSO: store in small aliquots at –80°C
  • Swelling buffer, ice cold (see recipe)
  • Energy mix (see recipe)
  • 15‐cm tissue culture plates (Corning, cat. no. 353025)
  • 2‐liter spinner flasks
  • Beckman Coulter J6‐M1 centrifuge and JS‐4.2 rotor for 250‐ to 500‐ml conical vessels
  • Sterile 20‐1/2G syringe needles
CAUTION: When working with human cells, all appropriate bio‐safety practices must be followed.NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic techniques should be used.
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Figures

Videos

Literature Cited

Literature Cited
  Ayad, N.G., Rankin, S., Ooi, D., Rape, M., and Kirschner, M.W. 2005. Identification of ubiquitin ligase substrates by in vitro expression cloning. Methods Enzymol. 399:404‐414.
  Del Rincon, S.V., Rogers, J., Widschwendter, M., Sun, D., Sieburg, H.B., and Spruck, C. 2010. Development and validation of a method for profiling post‐translational modification activities using protein microarrays. PLoS One 5:e11332.
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  Meng, L. 2012. Functional assays on high‐content protein microarrays. Curr. Protoc. Chem. Biol. 4:211‐231.
  Merbl, Y. and Kirschner, M.W. 2009. Large‐scale detection of ubiquitination substrates using cell extracts and protein microarrays. Proc. Natl. Acad. Sci. U.S.A. 106:2543‐2548.
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  Rape, M., Reddy, S.K., and Kirschner, M.W. 2006. The processivity of multiubiquitination by the APC determines the order of substrate degradation. Cell 124:89‐103.
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  Zhu, H., Bilgin, M., Bangham, R., Hall, D., Casamayor, A., Bertone, P., Lan, N., Jansen, R., Bidlingmaier, S., Houfek, T., Mitchell, T., Miller, P., Dean, R.A., Gerstein, M., and Snyder, M. 2001. Global analysis of protein activities using proteome chips. Science 293:2101‐2105.
Internet Resources
  http://www.invitrogen.com/site/us/en/home/Products‐and‐Services/Applications/Protein‐Expression‐and‐Analysis/Biomarker‐Discovery/ProtoArray.html
  This site describes the Human ProtoArray system and details of microarray production.
  http://www.invitrogen.com/site/us/en/home/Products‐and‐Services/Applications/Protein‐Expression‐and‐Analysis/Biomarker‐Discovery/ProtoArray/online‐tools.html
  This site describes online tools to analyze microarrays.
  http://www.mathworks.com/products/bioinfo/description3.html
  This site describes Bioinformatics Toolbox, a MATLAB add‐on that enables microarray data analysis.
  http://www.bioconductor.org/
  This site provides open source tools for large‐scale data analysis.
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