Proteomic Identification of Cellular Protease Substrates Using Isobaric Tags for Relative and Absolute Quantification (iTRAQ)

Richard A. Dean1, Derek Smith2, Christopher M. Overall1

1 University of British Columbia, Vancouver, British Columbia, Canada, 2 University of Victoria Proteomics Centre, Victoria, British Columbia, Canada
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 21.18
DOI:  10.1002/0471140864.ps2118s49
Online Posting Date:  August, 2007
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Identification of protease substrates is essential to identify and understand the functional consequences of normal and dysregulated proteolysis in disease on the proteome. Isobaric tags for relative and absolute quantification (iTRAQ) can be used to identify novel protease substrates in the cellular context. An amine‐targeted iTRAQ tag labels tryptic peptides generated from the proteins and protease cleavage products of secreted proteins, as well as protein domains shed from the cell membrane or pericellular matrix of protease‐transfected cells that have accumulated in conditioned medium; a second iTRAQ tag is used for control cells. MS/MS fragmentation enables sequencing of the pooled pairs of differently labeled but identical peptides and generates a low mass signature ion peak unique for each label. This signature ion peak identifies the peptides originating from the protease‐transfected or control cells; comparison of the peak areas enables relative quantitation of the peptide between the samples. Curr. Protoc. Protein Sci. 49:21.18.1‐21.18.12. © 2007 by John Wiley & Sons, Inc.

Keywords: iTRAQ; protease substrate identification; quantitative mass spectrometry

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Table of Contents

  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1:

  • Cells (see )
  • Cell culture medium (Invitrogen)
  • Phosphate buffer saline (PBS; appendix 2E)
  • Serum‐free, phenol‐red free culture medium (Invitrogen)
  • 0.5 mM phenylmethylsulfonyl fluoride (PMSF)
  • 1 mM EDTA
  • Appropriate broad‐spectrum inhibitor for the class of transfected protease
  • 100% acetonitrile (ACN)
  • 100% trifluoroacetic acid (TFA)
  • 50 mM HEPES, pH 8.0
  • 10% (w/v) sodium dodecyl sulfate (SDS)
  • 8 M deionized urea
  • Bicinchoninic acid (BCA) protein assay kit (or see unit 3.4)
  • Acetone
  • iTRAQ Reagents Multiplex Kit (Applied Biosystems)
  • Tris (2‐carboxyethyl) phosphine‐hydrochloride (TCEP)
  • Methyl methanethiosulfonate (MMTS)
  • Sequencing grade trypsin (Promega)
  • 0.5 M triethylammonium bicarbonate
  • KH 2PO 4, pH 2.7
  • 0.5 M KCl
  • HPLC solvent A: 2% ACN, 0.1% TFA
  • HPLC solvent B: 98% ACN, 0.1% TFA
  • Tissue culture flasks
  • 50‐ml tubes
  • Centrifuge that can accommodate up to 250‐ml bottles
  • 0.2‐µm pore size filtration unit
  • C4 and C18 3‐ml SPE cartridges (Grace VYDAC)
  • 1.5‐ml microcentrifuge tubes (Eppendorf)
  • SpeedVac concentrator
  • Sonicating bath
  • Heated lid temperature block
  • High performance liquid chromatography (HPLC) instrument
  • Polysulfoethyl A (Poly LC) 100‐mm × 4.6‐mm, 5‐µm‐300 Å strong cation exchange (SCX) column
  • Electrospray Qstar Pulsar mass spectometer (Applied Biosystems) or similar tandem mass spectrometer, with HPLC connected inline to permit analysis of samples directly from elution of columns
  • Analyst QS software (ABI/MDS SCIEX) or similar software
  • ProteinPilot (Applied Biosystems) or similar software
  • MASCOT search software (Matrix Science)
  • ProGroup viewer (Applied Biosystems), Microsoft Excel, or Access database
  • Software to match tandem mass spectra with peptide sequences (e.g., X! Tandem, ProteinPilot, or MASCOT)
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Literature Cited

Literature Cited
   Butler, G. and Overall, C.M. 2007. Proteomic validation of protease drug targets. Pharmacoproteomics of matrix metalloproteinase inhibitor drugs using isotope‐coded affinity tag labelling and tandem mass spectrometry. Curr. Pharm. Design 13:263‐270.
   Dean, R.A. and Overall, C.M. 2007. Proteomic discovery of metalloproteinase substrates in the cellular context by iTRAQ labeling reveals a diverse MMP‐2 substrate degradome. Mol. Cell Proteomics 6:611‐623.
   Lopez‐Otin, C. and Overall, C.M. 2002. Protease degradomics: A new challenge for proteomics. Nat. Rev. Mol. Cell Biol. 3:509‐519.
   Ross, P.L., Huang, Y.N., Marchese, J.N., Williamson, B., Parker, K., Hattan, S., Khainovski, N., Pillai, S., Dey, S., Daniels, S., Purkayastha, S., Juhasz, P., Martin, S., Bartlet‐Jones, M., He, F., Jacobson, A., and Pappin, D.J. 2004. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine‐reactive isobaric tagging reagents. Mol. Cell Proteomics 3:1154‐1169.
   Tam, E.M., Morrison, C.J., Wu, Y.I., Stack, M.S., and Overall, C.M. 2004. Membrane protease proteomics: Isotope‐coded affinity tag MS identification of undescribed MT1‐matrix metalloproteinase substrates. Proc. Natl. Acad. Sci. U.S.A. 101:6917‐6922.
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