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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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.
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