Peptide Separation Methodologies for In-depth Proteomics

  • Sajad Majeed Zargar
  • Rie Kurata
  • Randeep Rakwal
  • Yoichiro FukaoEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1242)


The integration of proteomics to other omics technologies and generation of proteome maps of a particular cell/tissue requires the identification and quantification of a maximum number of proteins. Traditional 2-D gel-based approach though provides a clear proteome map has its limitations, such as time consuming, requiring high skill, and most importantly, inability to identify low-abundance proteins. The most common drawback of 2-D gel electrophoresis is the masking of low amount proteins by the highly expressed (high abundance) proteins. Therefore, the elucidation of complete regulatory networks of a cell/tissue demands identification of low-abundance proteins. Low-abundance protein identification requires the use of usually gel-free mass spectrometry (MS)-based approaches. Using Arabidopsis thaliana as a model system, in this chapter, we describe all the steps followed for the extraction of microsomal proteins to MS analysis of separated peptides with a major focus on three different methods, namely, OFFGEL fractionation, 2D-LC, and long-column method for the identification of low-abundance proteins. Separation of such peptides will lead to in-depth proteomics-based investigations to answer biological questions.

Key words

Peptide fractionation Low-abundance proteins C-18 resin Peptide desalting MS 



This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas (No. 23119512 to Y.F.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan; a Grant-in-Aid for Scientific Research from Nara Institute of Science and Technology supported by The Ministry of Education, Culture, Sports, Science and Technology, Japan. This research was supported by Japan Advanced Plant Science Network. S.M.Z. acknowledges the DBT, New Delhi, India for award of CREST, Overseas postdoc fellowship. R.R. acknowledges the great support of Professors Yoshihiro Shiraiwa (Provost, Faculty of Life and Environmental Sciences, University of Tsukuba) and Koji Nomura (Organization for Educational Initiatives, University of Tsukuba) in promoting interdisciplinary research and unselfish encouragement.


  1. 1.
    Ahrens CH, Brunner E, Qeli E et al (2010) Generating and navigating proteome maps using mass spectrometry. Mol Cell Biol 11:789–801Google Scholar
  2. 2.
    Chen S, Harmon AC (2006) Advances in plant proteomics. Proteomics 6:5504–5516PubMedCrossRefGoogle Scholar
  3. 3.
    Chandramouli K, Qian PY (2009) Proteomics: challenges, techniques and possibilities to overcome biological sample complexity. Hum Genomics Proteomics 8:23920Google Scholar
  4. 4.
    Robertson D, Mitchell GP, Gilroy JS et al (1997) Differential extraction and protein sequencing reveals major differences in patterns of primary cell wall proteins from plants. J Biol Chem 272:15841–15848PubMedCrossRefGoogle Scholar
  5. 5.
    Molloy MP, Herbert BR, Walsh BJ et al (1998) Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis. Electrophoresis 19:837–844PubMedCrossRefGoogle Scholar
  6. 6.
    Borner GHH, Lilley KS, Stevens TJ et al (2003) Identification of glycosylphosphatidylinositol-anchored proteins in Arabidopsis: a proteomic and genomic analysis. Plant Physiol 132:568–577PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Peltier JB, Ytterberg AJ, Sun Q et al (2004) New functions of the thylakoid membrane proteome of Arabidopsis thaliana revealed by a simple, fast, and versatile fractionation strategy. J Biol Chem 279:49367–49383PubMedCrossRefGoogle Scholar
  8. 8.
    Brown JWS, Flavell RB (1981) Fractionation of wheat gliadin and glutenin subunits by two-dimensional electrophoresis and the role of group 6 and group 2 chromosomes in gliadin synthesis. Theor Appl Genet 59:349–359PubMedGoogle Scholar
  9. 9.
    Werhahn W, Braun HP (2002) Biochemical dissection of the mitochondrial proteome from Arabidopsis thaliana by three-dimensional gel electrophoresis. Electrophoresis 23:640–646PubMedCrossRefGoogle Scholar
  10. 10.
    Heazlewood JL, Tonti-Filippini JS, Gout AM et al (2004) Experimental analysis of the Arabidopsis mitochondrial proteome highlights signaling and regulatory components, provides assessment of targeting prediction programs, and indicates plant-specific mitochondrial proteins. Plant Cell 16:241–256PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Bae MS, Cho EJ, Choi EY et al (2003) Analysis of the Arabidopsis nuclear proteome and its response to cold stress. Plant J 36:652–663PubMedCrossRefGoogle Scholar
  12. 12.
    Pendle AF, Clark GP, Boon R et al (2005) Proteomic analysis of the Arabidopsis nucleolus suggests novel nucleolar functions. Mol Biol Cell 16:260–269PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Carter C, Pan S, Zouhar J et al (2004) The vegetative vacuole proteome of Arabidopsis thaliana reveals predicted and unexpected proteins. Plant Cell 16:3285–3303PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Szponarski W, Sommerer N, Boyer JC et al (2004) Large-scale characterization of integral proteins from Arabidopsis vacuolar membrane by two-dimensional liquid chromatography. Proteomics 4:397–406PubMedCrossRefGoogle Scholar
  15. 15.
    Fukao Y, Hayashi M, Nishimura M (2002) Proteomic analysis of leaf peroxisomal proteins in greening cotyledons of Arabidopsis thaliana. Plant Cell Physiol 43:689–696PubMedCrossRefGoogle Scholar
  16. 16.
    Santoni V, Kieffer S, Desclaux D et al (2000) Membrane proteomics: use of additive main effects with multiplicative interaction model to classify plasma membrane proteins according to their solubility and electrophoretic properties. Electrophoresis 21:3329–3344PubMedCrossRefGoogle Scholar
  17. 17.
    Alexandersson E, Saalbach G, Larsson C et al (2004) Arabidopsis plasma membrane proteomics identifies components of transport, signal transduction and membrane trafficking. Plant Cell Physiol 45:1543–1556PubMedCrossRefGoogle Scholar
  18. 18.
    Chivasa S, Ndimba BK, Simon WJ et al (2002) Proteomic analysis of the Arabidopsis thaliana cell wall. Electrophoresis 23:1754–1765PubMedCrossRefGoogle Scholar
  19. 19.
    Haslam RP, Downie AL, Raveton M et al (2003) The assessment of enriched apoplastic extracts using proteomic approaches. Ann Appl Biol 143:81–91CrossRefGoogle Scholar
  20. 20.
    Agrawal GK, Bourguignon J, Rolland N et al (2011) Plant organelle proteomics: collaborating for optimal cell function. Mass Spectrom Rev 30:772–853PubMedGoogle Scholar
  21. 21.
    Agrawal GK, Rakwal R (2008) Plant proteomics: technologies, strategies, and applications. Wiley, HobokenCrossRefGoogle Scholar
  22. 22.
    Bjellqvist B, Hughes GJ, Pasquali C et al (1993) The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences. Electrophoresis 14:1023–1031PubMedCrossRefGoogle Scholar
  23. 23.
    Horth P, Miller CA, Prekel T et al (2006) Efficient fractionation and improved protein identification by peptide OFFGEL electrophoresis. Mol Cell Proteomics 5:1968–1974PubMedCrossRefGoogle Scholar
  24. 24.
    Chenau J, Michelland S, Sidibe J et al (2008) Peptides OFFGEL electrophoresis: a suitable pre-analytical step for complex eukaryotic samples fractionation compatible with quantitative iTRAQ labeling. Proc Natl Acad Sci U S A 6:1–8Google Scholar
  25. 25.
    Fraterman S, Zeiger U, Khurana TS et al (2007) Combination of peptide OFFGEL fractionation and label-free quantitation facilitated proteomics profiling of extraocular muscle. Proteomics 7:3404–3416PubMedCrossRefGoogle Scholar
  26. 26.
    Abdallah C, Sergeant K, Guillier C et al (2012) Optimization of iTRAQ labelling coupled to OFFGEL fractionation as a proteomic workflow to the analysis of microsomal proteins of Medicago truncatula roots. Proc Natl Acad Sci U S A 10:37Google Scholar
  27. 27.
    Cargile BJ, Sevinsky JR, Essader AS et al (2005) Immobilized pH gradient isoelectric focusing as a first-dimension separation in shotgun proteomics. J Biomol Tech 16:181–189PubMedCentralPubMedGoogle Scholar
  28. 28.
    Wolters DA, Washburn MP, Yates JR (2001) An automated multidimensional protein identification technology for shotgun proteomics. Anal Chem 73:5683–5690PubMedCrossRefGoogle Scholar
  29. 29.
    Gilar M, Olivova P, Daly AD et al (1995) Two-dimensional separation of peptides using RP-RP-HPLC system with different pH in first and second separation dimensions. J Sep Sci 28:1694–1703CrossRefGoogle Scholar
  30. 30.
    Miyamoto K, Hara T, Kobayashi H et al (2008) High-efficient liquid chromatographic separation uitilising long monolithic silica capillary columns. Anal Chem 80:8741–8750PubMedCrossRefGoogle Scholar
  31. 31.
    Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco cultures. Plant Physiol 15:473–497CrossRefGoogle Scholar
  32. 32.
    Fukao Y, Ferjani A, Fujiwara M et al (2009) Identification of zinc-responsive proteins in the roots of Arabidopsis thaliana using a highly improved method of two-dimensional electrophoresis. Plant Cell Physiol 50:2234–2239PubMedCrossRefGoogle Scholar
  33. 33.
    Fukao Y, Ferjani A, Tomioka R et al (2011) iTRAQ analysis reveals mechanisms of growth defects due to excess zinc in Arabidopsis. Plant Physiol 155:1893–1907PubMedCentralPubMedCrossRefGoogle Scholar
  34. 34.
    Fukao Y, Yoshida M, Kurata R et al (2013) Peptide separation methodologies for in-depth proteomics in Arabidopsis. Plant Cell Physiol 54(5):808–815PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Sajad Majeed Zargar
    • 1
    • 2
  • Rie Kurata
    • 1
  • Randeep Rakwal
    • 3
  • Yoichiro Fukao
    • 1
    • 4
    Email author
  1. 1.Graduate School of Biological SciencesNara Institute of Science and TechnologyIkomaJapan
  2. 2.School of BiotechnologySK University of Agricultural Sciences and TechnologyJammuIndia
  3. 3.Organization for Educational InitiativesUniversity of TsukubaTsukubaJapan
  4. 4.Plant Global Educational ProjectNara Institute of Science and TechnologyIkomaJapan

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