Advertisement

Plant Molecular Biology Reporter

, Volume 31, Issue 1, pp 47–54 | Cite as

An Efficient In-gel Digestion Protocol for Mass Spectral Analysis by MALDI-TOF-MS and MS/MS and Its Use for Proteomic Analysis of Vigna mungo Leaves

  • Subrata Kundu
  • Dipjyoti Chakraborty
  • Kalipada Das
  • Amita PalEmail author
Original Paper

Abstract

An efficient protocol for in-gel digestion of Coomassie-stained protein spots has been established for mass analysis by matrix-assisted laser desorption/ionization-mass spectrometry (MS) and for tandem mass spectrometry (MS/MS). Identification of Vigna mungo leaf proteome from two-dimensional gel electrophoresis was done employing the protocol. About 300 proteins spots were consistently detected in three replicate gels. Optimization of the destaining process, digestion using 25 ng/μl trypsin in 20 μl trypsin buffer, and omission of peptide extraction step significantly increased the number of matched peptides and sequence coverage. Reliable characterization of 109 proteins by MS as well as tandem sequencing by MS/MS (PRIDE Accession no. 15318) suggests the potential application of the modified protocol for high throughput proteome analysis to unravel disputes in characterization of plant proteins in fundamental or applied research.

Keywords

In-gel digestion MALDI-TOF-MS and MS/MS Plant proteomics Vigna mungo 

Notes

Acknowledgments

The authors thank the Department of Biotechnology, India, for the financial assistance (BT/01/COE/06/03) and research fellowships to D.C. and S.K., and the Director, Bose Institute, for providing lab facilities. The proteomic facilities provided by DST through IRHPA project (IR/SO/LF02/2002) and SERC project (SR/SO/PS-58/05) are also thankfully acknowledged.

Supplementary material

11105_2012_475_MOESM1_ESM.pdf (815 kb)
ESM 1 [(PDF 814 kb)

References

  1. Aghaei K, Ali AE, Setsuko K (2008) Proteome analysis of potato under salt stress. J Proteome Res 7:4858–4868PubMedCrossRefGoogle Scholar
  2. Basak J, Kundagrami S, Ghosh TK, Pal A (2005) Development of yellow mosaic virus (YMV) resistance linked DNA marker in Vigna mungo from populations segregating for YMV-reaction. Mol Breed 14:375–383CrossRefGoogle Scholar
  3. Basha SM, Roberts RM (1981) The glycoproteins of plant seeds: analysis by two-dimensional polyacrylamide gel electrophoresis and by their lectin-binding properties. Plant Physiol 67:936–939PubMedCrossRefGoogle Scholar
  4. Basha SM, Katam R, Vasanthaiah H, Matta F (2009) Proteome analysis of muscadine grape leaves. Int J Wine Res 1:1–13Google Scholar
  5. Bradford MM (1976) A rapid and sensitive method for quantification of proteins utilizing the principle of protein dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  6. Ding C, You J, Liu Z, Rehmani IA, Wang S, Li G, Wang O, Ding Y (2011) Proteomic analysis of low nitrogen stress-responsive proteins in roots of rice. Plant Mol Biol Rep 29:618–625CrossRefGoogle Scholar
  7. Emanuelsson O, Brunak S, Heijne G, Nielsen H (2007) Locating proteins in the cell using TargetP, SignalP and related tools. Nat Protoc 2:953–971PubMedCrossRefGoogle Scholar
  8. Granvogl B, Ploscher M, Eichacker LA (2007) Sample preparation by in-gel digestion for mass spectrometry-based proteomics. Anal Bioanal Chem 389:991–1002PubMedCrossRefGoogle Scholar
  9. Hashiguchi A, Sakata K, Komatsu S (2009) Proteome analysis of early-stage soybean seedlings under flooding stress. J Proteome Res 8:2058–2069PubMedCrossRefGoogle Scholar
  10. Havlis J, Thomas H, Sebela M, Shevchenko A (2003) Fast-response proteomics by accelerated in-gel digestion of proteins. Anal Chem 75:1300–1306PubMedCrossRefGoogle Scholar
  11. Isaacson T, Damasceno CMB, Saravanan RS, He Y, Catala C, Saladie M, Rose JKC (2006) Sample extraction techniques for enhanced proteomic analysis of plant tissues. Nat Protoc 1:769–774PubMedCrossRefGoogle Scholar
  12. Kong F, Mao S, Jiang J, Wang J, Fang X, Wang Y (2011) Proteomic changes in newly synthesized Brassica napus allotetraploids and their early generations. Plant Mol Biol Rep 29:927–935CrossRefGoogle Scholar
  13. Kumarathasan P, Mohottalage S, Goegan P, Vincent R (2005) An optimized protein in-gel digest method for reliable proteome characterization by MALDI-TOF-MS analysis. Anal Biochem 346:85–89PubMedCrossRefGoogle Scholar
  14. Kundagrami S, Basak J, Maiti S, Kundu A, Das B, Ghosh TK, Pal A (2009) Agronomic, genetic and molecular characterization of MYMIV tolerant mutant lines of Vigna mungo. Int J Plant Breed Genet 3:1–10CrossRefGoogle Scholar
  15. Kundu S, Chakraborty D, Pal A (2011) Proteomic analysis of salicylic acid induced resistance to Mungbean Yellow Mosaic India Virus in Vigna mungo. J Proteomics 74:337–349PubMedCrossRefGoogle Scholar
  16. Lin H, Tseng L (2010) DBCP: a web server for disulfide bonding connectivity pattern prediction without the prior knowledge of the bonding state of cysteines. Nucleic Acids Res 38(suppl 2):W503–W507PubMedCrossRefGoogle Scholar
  17. Shen S, Yuxiang J, Kuang T (2003) Proteomics approach to identify wound-response related proteins from rice leaf sheath. Proteomics 3:527–535PubMedCrossRefGoogle Scholar
  18. Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M (2007) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1:2856–2860CrossRefGoogle Scholar
  19. Speicher KD, Kolbas O, Harper S, Speicher DW (2000) Systematic analysis of peptide recoveries from in-gel digestions for protein identifications in proteome studies. J Biomol Tech 11:74–86PubMedGoogle Scholar
  20. Yin H, Yan F, Ji J, Li Y, Wang R, Xu C (2012) Proteomic analysis of Arabidopsis thaliana leaves infested by tobacco whitefly Bemisia tabaci (Gennadius) B biotype. Plant Mol Biol Rep 30:379–390CrossRefGoogle Scholar
  21. Zhou G, Yang LT, Li YR, Zou CL, Huang LP, Qiu LH, Huang X, Srivastava MK (2012) Proteomic analysis of osmotic stress-responsive proteins in sugarcane leaves. Plant Mol Biol Rep 30:349–359CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Subrata Kundu
    • 1
  • Dipjyoti Chakraborty
    • 1
    • 2
  • Kalipada Das
    • 3
  • Amita Pal
    • 1
    Email author
  1. 1.Division of Plant BiologyBose InstituteKolkataIndia
  2. 2.Department of Bioscience and BiotechnologyBanasthali VidyapithRajasthanIndia
  3. 3.Department of ChemistryBose InstituteKolkataIndia

Personalised recommendations