Experimental and Computational Tools for Analysis of Signaling Networks in Primary Cells

Erwin M. Schoof1, Rune Linding1

1 Cellular Signal Integration Group (C‐SIG), Center for Biological Sequence Analysis (CBS), Department of Systems Biology, Technical University of Denmark (DTU), Lyngby
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 11.11
DOI:  10.1002/0471142735.im1111s104
Online Posting Date:  February, 2014
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Cellular information processing in signaling networks forms the basis of responses to environmental stimuli. At any given time, cells receive multiple simultaneous input cues, which are processed and integrated to determine cellular responses such as migration, proliferation, apoptosis, or differentiation. Protein phosphorylation events play a major role in this process and are often involved in fundamental biological and cellular processes such as protein‐protein interactions, enzyme activity, and immune responses. Determining which kinases phosphorylate specific phospho sites poses a challenge; this information is critical when trying to elucidate key proteins involved in specific cellular responses. Here, methods to generate high‐quality quantitative phosphorylation data from cell lysates originating from primary cells, and how to analyze the generated data to construct quantitative signaling network models, are presented. These models can subsequently be used to guide follow‐up in vitro/in vivo validation studies. Curr. Protoc. Immunol. 104:11.11.1‐11.11.23. © 2014 by John Wiley & Sons, Inc.

Keywords: phosphorylation; mass spectrometry; network biology; primary cell signaling

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Table of Contents

  • Cell Signaling in Primary Cells
  • Generating Quantitative Phospho‐Proteomics Data Using Mass Spectrometry
  • Basic Protocol 1: Cell Lysis, Protein Digestion, and Dimethyl Labeling
  • Basic Protocol 2: SCX Fractionation
  • Basic Protocol 3: Titanium Dioxide Phosphopeptide Enrichment
  • Basic Protocol 4: Mass Spectrometry Sample Preparation
  • Basic Protocol 5: Analyzing Phosphorylation Data and Constructing Quantitative Network Models
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Cell Lysis, Protein Digestion, and Dimethyl Labeling

  • Cell line(s) of interest
  • Phosphate‐buffered saline (PBS; Sigma, cat. no. P5368), ice cold
  • Modified RIPA buffer (see recipe), ice cold
  • Acetone, HPLC‐grade (Sigma, cat. no. 650501), −20°C
  • Denaturation buffer (see recipe)
  • Bradford reagent (Sigma, cat. no. B6916)
  • Dithiothreitol (DTT; Sigma, cat. no. 43815)
  • Chloroacetamide (CAA; Sigma, cat. no. 22790)
  • Lysyl endopeptidase (Lys‐C; Wako, cat. no. 129‐02541; 0.5 µg/µl stock solution made up in MilliQ water)
  • Triethyl ammonium bicarbonate (TEAB; Sigma, cat. no. T7408)
  • Trypsin (Sigma, cat. no. T6567; 0.5 µg/µl stock solution made up in 50 mM acetic acid)
  • Trifluoroacetic acid (TFA; Sigma, cat. no. T6508)
  • Acetic acid (Fisher Scientific, cat. no. A35‐500)
  • Dimethyl labeling solution (see recipe)
  • 15‐ or 50‐ml tubes
  • Sonicator
  • Refrigerated centrifuge
  • Axial rotator
  • SepPak C18 columns (Waters, cat. no. WAT020515)
  • 10‐ml syringe (polypropylene)
  • Additional reagents and equipment for Bradford assay (Bradford, )

Basic Protocol 2: SCX Fractionation

  • Sample
  • Acetonitrile, HPLC‐grade (Sigma, cat. no. 34851N)
  • SCX buffer A (see recipe)
  • SCX buffer B (see recipe)
  • Loading buffer: 1%TFA and 2% acetonitrile in MS H 2O
  • HPLC/FPLC system (e.g., GE Healthcare AktaMicro)
  • 1‐ml SCX column or equivalent (e.g., Resource S 1ml; GE Healthcare Resources)
  • 2‐ml microcentrifuge tubes

Basic Protocol 3: Titanium Dioxide Phosphopeptide Enrichment

  • TiO 2 beads (GL Sciences, cat. no. 5020‐75010)
  • TiO 2 loading solution (see recipe)
  • SCX samples (see protocol 2)
  • SCX buffer B (see recipe)
  • TiO 2 washing solution 1 (see recipe)
  • TiO 2 washing solution 2 (see recipe)
  • Acidification buffer (see recipe)
  • TiO 2 elution buffer 1 (see recipe)
  • TiO 2 elution buffer 2 (see recipe)
  • Automated sample shaker (e.g., Eppendorf Thermomixer)
  • End‐over‐end rotator
  • Centrifuge
  • C8 StageTips (Thermo Fisher, cat. no. SP321)
  • 10‐ml luer‐lock syringes and StageTip adaptor (Millian, cat. no. HAM‐31330)
  • 96‐well PCR plates
  • Vacuum centrifuge with microplate rotor (e.g., Thermo Savant SC250)
  • Litmus paper
  • Vortex

Basic Protocol 4: Mass Spectrometry Sample Preparation

  • Methanol, HPLC‐grade (Sigma, cat. no. 34860)
  • Buffer B (see recipe)
  • Sample buffer (see recipe)
  • Samples (see Basic Protocol protocol 11, protocol 22, or protocol 33)
  • Buffer A (see recipe)
  • Loading buffer (see recipe)
  • C18 StageTips (Thermo Fisher, cat. no. SP301)
  • 10‐ml luer‐lock syringes and StageTip adaptors (Millian, cat. no. HAM‐31330)
  • Vacuum centrifuge (e.g., Thermo Savant SC250)
  • Mass spectrometer with nanospray source (e.g., Thermo Fisher Q Exactive or Orbitrap Fusion)

Basic Protocol 5: Analyzing Phosphorylation Data and Constructing Quantitative Network Models

  • Desktop computer with Internet access
  • Mass spectrometry spectral matching software (e.g., MaxQuant or Proteome Discoverer/SEQUEST)
  • R statistical software
  • Visual network editing software (e.g., Gephi.org or Cytoscape.org)
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Internet Resources
  Internet portal to access the integrated NetPhorest and NetworKIN frameworks.
  Internet portal to access the original NetworKIN framework.
  Internet portal to access the original NetPhorest framework.
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