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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

Plant Molecular Biology Reporter volume 31, pages 47–54 (2013)Cite this article

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.

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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.

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Authors and Affiliations

  1. Division of Plant Biology, Bose Institute, Centenary Campus, P 1/12, C. I. T. Road, Scheme VIIM, Kolkata, 700054, West Bengal, India

    Subrata Kundu, Dipjyoti Chakraborty & Amita Pal

  2. Department of Bioscience and Biotechnology, Banasthali Vidyapith, Rajasthan, 304022, India

    Dipjyoti Chakraborty

  3. Department of Chemistry, Bose Institute, Kolkata, 700009, WB, India

    Kalipada Das

Authors
  1. Subrata Kundu
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  2. Dipjyoti Chakraborty
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  3. Kalipada Das
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  4. Amita Pal
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Corresponding author

Correspondence to Amita Pal.

Additional information

Subrata Kundu and Dipjyoti Chakraborty contributed equally to this paper.

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Kundu, S., Chakraborty, D., Das, K. et al. 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. Plant Mol Biol Rep 31, 47–54 (2013). https://doi.org/10.1007/s11105-012-0475-x

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  • DOI: https://doi.org/10.1007/s11105-012-0475-x

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