Plant iTRAQ‐Based Proteomics

Pubudu P. Handakumbura1, Kim K. Hixson1, Samuel O. Purvine1, Christer Jansson1, Ljiljana Paša‐Tolić1

1 Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20052
Online Posting Date:  June, 2017
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Abstract

We present a simple one‐pot extraction protocol, which rapidly isolates hydrophilic metabolites, lipids, and proteins from the same pulverized plant sample. Also detailed is a global plant proteomics sample preparation method utilizing iTRAQ multiplexing reagents that enables deep proteome coverage due to the use of HPLC fractionation of the peptides prior to mass spectrometric analysis. We have successfully used this protocol on several different plant tissues (e.g., roots, stems, leaves) from different plants (e.g., sorghum, poplar, Arabidopsis, soybean), and have been able to successfully detect and quantify thousands of proteins. Multiplexing strategies such as iTRAQ and the bioinformatics strategy outlined here, ultimately provide insight into which proteins are significantly changed in abundance between two or more groups (e.g., control, perturbation). Our bioinformatics strategy yields z‐score values, which normalize the expression data into a format that can easily be cross‐compared with other expression data (i.e., metabolomics, transcriptomics) obtained from different analytical methods and instrumentation. © 2017 by John Wiley & Sons, Inc.

Keywords: iTRAQ; plant; sorghum; quantitative proteomics

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Single‐Pot Extraction Protocol
  • Basic Protocol 2: Detailed Global Proteomics Protocol
  • Support Protocol 1: C‐18 Solid Phase Extraction Cleanup
  • Support Protocol 2: iTRAQ 8‐Plex Labeling
  • Support Protocol 3: Strong Cation Exchange Cleanup Method
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Single‐Pot Extraction Protocol

  Materials
  • Flash frozen tissue samples
  • Liquid nitrogen
  • Methanol (stored at −20°C)
  • Chloroform (stored at −20°C)
  • Nanopure water
  • Mortar and pestle, tissue lyzer (e.g., for soft plant tissues such as leaves), freezer mill (for difficult to pulverize samples such as stems and roots), or kitchen blender
  • Spatulas
  • Analytical balance
  • 15‐ml tubes (to perform extraction; compatible with chloroform, Genesee Scientific, cat. no. 21‐103)
  • Centrifuge with temperature regulator
  • Glass Pasteur pipets (chloroform should always be dispensed and handled with glass)
  • Glass v‐vials or Sorenson MulTI SafeSeal microcentrifuge tubes
  • Autosampler vials (MicroSolv Technology Corporation, cat. no. 9502 S‐WCV) with caps (MicroSolv Technology Corporation, cat. no. 9502 S‐20 C‐B‐M; lipid and metabolite fractions should be collected with these)

Basic Protocol 2: Detailed Global Proteomics Protocol

  Materials
  • Dried protein pellets or pulverized flash frozen tissue sample
  • 0.1 M ammonium acetate in methanol, ice cold
  • β‐mercaptoethanol
  • Coomassie Plus assay kit (Thermo Fisher Scientific, cat. no. PI23238)
  • Bovine serum albumin (BSA) standard, 2 mg/ml (Thermo Fisher Scientific, cat. no. 23209)
  • Protein solubilization buffer (see recipe)
  • 1 M chloroacetamide
  • 50 mM ammonium bicarbonate (freshly made)
  • 1 M CaCl 2
  • 1 mg/ml trypsin (mass spectrometry grade, Affymetrix, cat. no. 22725)
  • 10% trifluoroacetic acid (TFA)
  • BCA assay kit (Thermo Fisher Scientific, cat. no. PI23227)
  • iTRAQ 8‐plex reagent kit (Sciex, cat. no. 4393529)
  • High pH HPLC Solvent A (10 mM ammonium formate, adjusted to pH 10 using ammonium hydroxide)
  • High pH HPLC Solvent B (10 mM ammonium formate, 90% acetronitrile, adjusted to pH 10 using ammonium hydroxide)
  • Acetonitrile
  • Nanopure water
  • 2‐ml individually wrapped siliconized microcentrifuge tubes
  • Centrifuge
  • Serological pipets
  • Plate reader with wavelengths 562 and 595 nm
  • Sonicator
  • Vortexer
  • Single‐step filter vials (Thomas Instrument Company, cat. no. 33540‐100)
  • C‐18 SPE cleanup columns (Phenomenex, cat. no. 8B‐S001‐DAK) and vacuum chamber
  • Speed Vac
  • Auto sampler vials
  • Agilent HPLC 1100 series with XBridge C18 column, 250 × 4.6 mm, 5 μM with 4.6 × 20 mm guard column (Waters)

Support Protocol 1: C‐18 Solid Phase Extraction Cleanup

  Materials
  • Methanol
  • C‐18 rinse solution (0.1% TFA in water)
  • Peptide samples
  • C‐18 elution solution (80:20 acetonitrile/0.1% TFA in water)
  • C‐18 SPE cleanup columns (Phenomenex, cat. no. 8B‐S001‐DAK)
  • Speed Vac

Support Protocol 2: iTRAQ 8‐Plex Labeling

  Materials
  • 8‐plex iTRAQ reagent kit (Sigma‐Aldrich, contains all buffers referenced here)
  • Dried peptide samples
  • 50 mM ammonium bicarbonate
  • 2‐ml siliconized microcentrifuge tubes
  • Speed Vac

Support Protocol 3: Strong Cation Exchange Cleanup Method

  Materials
  • Methanol
  • 1% TFA in water
  • Wash solvent (0.1% TFA in water)
  • Rinse solvent (7:3 methanol/0.1% TFA in water)
  • Peptide samples
  • 7:3 (v/v) 5% NH 4OH/methanol
  • SCX SPE extraction columns (Sigma Discovery)
  • Speed Vac
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Figures

Videos

Literature Cited

Literature Cited
   Bantscheff, M. , Boesche, M. , Eberhard, D. , Matthieson, T. , Sweetman, G. , & Kuster, B. (2008). Robust and sensitive iTRAQ quantification on an LTQ orbitrap mass spectrometer. Molecular & Cellular Proteomics, 7, 1702–1713. doi: 10.1074/mcp.M800029‐MCP200.
   Chen, L. H. , Huang, Y. N. , Xu, M. , Cheng, Z. X. , Zhang, D. S. , & Zheng, J. G. (2016). iTRAQ‐based quantitative proteomics analysis of black rice grain development reveals metabolic pathways associated with anthocyanin biosynthesis. Plos One, 11, e015923. doi: 10.1371/journal.pone.0159238.
   Gygi, S. P. , Rist, B. , Gerber, S. A. , Turecek, F. , Gelb, M. H. , & Aebersold, R. (1999). Quantitative analysis of complex protein mixtures using isotope‐coded affinity tags. Nature Biotechnology, 17, 994‐999. doi: 10.1038/13690.
   Karp, N. A. , Huber, W. , Sadowski, P. G. , Charles, P. D. , Hester, S. V. , & Lilley, K. S. (2010). Addressing accuracy and precision issues in iTRAQ quantitation. Molecular & Cellular Proteomics, 9, 1885–1897. doi: 10.1074/mcp.M900628‐MCP200.
   Li, Q. , Chang, R. , Sun, Y. , & Li, B. (2016). iTRAQ‐based quantitative proteomic analysis of Spirulina platensis in response to low temperature stress. PLoS One, 11, e0166876. doi: 10.1371/journal.pone.0166876.
   Love, M. I. , Huber, W. , & Anders, S. (2014). Moderated estimation of fold change and dispersion for RNA‐seq data with DESeq2. Genome Biology, 15, 550. doi: 10.1186/s13059‐014‐0550‐8.
   Nakayasu, E. S. , Brown, R. N. , Ansong, C. , Sydor, M. A. , Imtiaz, S. , Mihai, C. , … Adkins, J. N. (2013). Multi‐omic data integration links deleted in breast cancer 1 (DBC1) degradation to chromatin remodeling in inflammatory response. Molecular & Cellular Proteomics, 12, 2136‐2147. doi: 10.1074/mcp.M112.026138.
   Nguyen, T. H. , Brechenmacher, L. , Aldrich, J. T. , Clauss, T. R. , Gritsenko, M. A. , Hixson, K. K. , … Stacey, G. (2012). Quantitative phosphoproteomic analysis of soybean root hairs inoculated with Bradyrhizobium japonicum . Molecular & Cellular Proteomics, 11, 1140–1155. doi: 10.1074/mcp.M112.018028.
   Robinson, M. D. , McCarthy, D. J. , & Smyth, G. K. (2009). edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics, 26, 136–140. doi: 10.1093/bioinformatics/btp616.
   Tabb, D. L. , Wang, X. , Carr, S. A. , Clauser, K. R. , Mertins, P. , Chambers, M. C. , … Liebler, D. C. (2016). Reproducibility of differential proteomic technologies in CPTAC fractionated xenografts. Journal of Proteome Research, 15, 691–706. doi: 10.1021/acs.jproteome.5b00859.
   Thompson, A. , Schafer, J. , Kuhn, K. , Kienle, S. , Schwarz, J. , Schmidt, G. , … Hamon, C. (2003). Tandem mass tags: A novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Analytical Chemistry, 75, 1895–1904. doi: 10.1021/ac0262560.
   Wang, B. A. , Hajano, J. U. D. , Ren, Y. D. , Lu, C. T. , & Wang, X. F. (2015). iTRAQ‐based quantitative proteomics analysis of rice leaves infected by Rice stripe virus reveals several proteins involved in symptom formation. Virology Journal, 12, 99. doi: 10.1186/s12985‐015‐0328‐y.
   Wiese, S. , Reidegeld, K. A. , Meyer, H. E. , & Warscheid, B. (2007). Protein labeling by iTRAQ: A new tool for quantitative mass spectrometry in proteome research. Proteomics, 7, 340–350. doi: 10.1002/pmic.200600422.
   Yang, F. , Shen, Y. , Camp, D. G. , & Smith, R. D. (2012). High‐pH reversed‐phase chromatography with fraction concatenation for 2D proteomic analysis. Expert Review of Proteomics, 9, 129–134. doi: 10.1586/epr.12.15.
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