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


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.


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



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)


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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

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