Analysis of Serine‐Threonine Kinase Specificity Using Arrayed Positional Scanning Peptide Libraries

Catherine Chen1, Benjamin E. Turk1

1 Yale University, New Haven, Connecticut
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 18.14
DOI:  10.1002/0471142727.mb1814s91
Online Posting Date:  July, 2010
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Protein kinases vary substantially in their consensus phosphorylation motifs, the residues that are either preferred or deselected by the kinase at specific positions surrounding the phosphorylation site. The protocol described here is used to rapidly determine phosphorylation motifs for serine‐threonine kinases. The procedure involves screening an arrayed combinatorial peptide library consisting of 198 biotinylated substrates. Peptides are phosphorylated by the kinase of interest in the presence of radiolabeled ATP and then captured on streptavidin membrane. The membrane is subsequently washed, dried, and exposed to a phosphor screen to visualize and quantify incorporation of radiolabel into the peptides. The phosphorylation motif is thereby derived from the relative extent of phosphorylation of each peptide in the array. Curr. Protoc. Mol. Biol. 91:18.14.1‐18.14.15. © 2010 by John Wiley & Sons, Inc.

Keywords: phosphorylation motif; kinase specificity; peptide array

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Peptide Array Screening in 384‐Well Plates
  • Alternate Protocol 1: Peptide Array Screening in 1536‐Well Plates
  • Support Protocol 1: Preparation of Peptide Stock Solutions and Plates
  • Support Protocol 2: Washing and Imaging of Peptides Bound to Streptavidin Membrane
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Peptide Array Screening in 384‐Well Plates

  • Ser/Thr peptide library set (Anaspec, cat. no. 62017): 0.6 mM aqueous stocks arrayed in a 384‐well plate (see protocol 3)
  • Kinase reaction buffer (see recipe)
  • 0.1% Tween 20
  • Isopropanol
  • 4× kinase solution (typically 10 to 50 µg/ml, diluted fresh in kinase reaction buffer and kept on ice)
  • 4× ATP solution (200 µM cold ATP/0.1 µCi/µl [γ‐33P]ATP or [γ‐32P]ATP, diluted in kinase reaction buffer and kept on ice)
  • SAM2 streptavidin membrane (Promega)
  • SDS wash buffer (see recipe)
  • 20‐µl multichannel pipettor
  • 384‐well clear polystyrene plates
  • Centrifuge with microplate carriers
  • Alignment frames for slot pin replicator (V&P Scientific, cat. no. VP381 Library Copier)
  • 384‐slot pin replicators (2‐µl volume; V&P Scientific, cat. no. VP384S2)
  • Reagent reservoirs for the slot pin replicator
  • Lint‐free blotting paper (V&P Scientific)
  • Clear adhesive seals for multi‐well plates and plate sealer (optional but recommended)
  • 30°C incubator
  • 4 × 6–in. rubber mat (available from art supply store)

Alternate Protocol 1: Peptide Array Screening in 1536‐Well Plates

  • Aluminum adhesive (Corning Costar, cat. no. 6570)
  • 10× kinase/ATP solution (see recipe)
  • Pin cleaning solution (V&P Scientific)
  • Clear polystyrene 1536‐well plates
  • 1536‐well slot pin replicators with 5 rows of 48 floating 200‐nl pins (V&P Scientific, cat. no. FP3S200)
  • Pin‐tool strip with one row of 48 floating 200‐nl pins (V&P Scientific)
  • Alignment frames with four pairs of guide holes for 1536‐well replicator (V&P Scientific)
  • Alignment frame with guide holes for each row of a 1536‐well plate for the pin tool strip (V&P Scientific)

Support Protocol 1: Preparation of Peptide Stock Solutions and Plates

  • 180‐member peptide library, 1 mg per peptide (Anaspec, cat. no. 62017‐1)
  • 18‐member phosphopeptide library, 1 mg per peptide (Anaspec, cat. no. 62335)
  • DMSO, degassed by bubbling with argon for 5 min
  • 20 mM HEPES, pH 7.4
  • Spectrophotometer
  • 0.5‐ or 1.5‐ml polypropylene microcentrifuge tubes
  • Multi‐well storage plates, either 384‐well polypropylene plates (for protocol 1) or 1536‐well polystyrene plates (for protocol 2)
  • Aluminum multi‐well plate seals suitable for storage at −20°C (Corning Costar, cat. no. 6570)

Support Protocol 2: Washing and Imaging of Peptides Bound to Streptavidin Membrane

  • Streptavidin membrane strip from protocol 1 or protocol 2
  • SDS wash buffer (see recipe)
  • 2 M NaCl
  • 2 M NaCl/1% H 3PO 4
  • Benchtop orbital or rocking platform shaker
  • Aluminum foil
  • Plastic wrap
  • Storage phosphor system with image analysis software (BioRad Personal Molecular Imager with ImageQuant software or the equivalent)
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Literature Cited

Literature Cited
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