Rapid mass spectrometric determination of disulfide connectivity in peptides and proteins

Bhattacharyya, Moitrayee and Gupta, Kallol and Gowd, Konkallu Hanumae and Balaram, Padmanabhan (2013) Rapid mass spectrometric determination of disulfide connectivity in peptides and proteins. In: Molecular BioSystems, 9 (6). pp. 1340-1350.

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Abstract

Disulfide crosslinks are ubiquitous in natural peptides and proteins, providing rigidity to polypeptide scaffolds. The assignment of disulfide connectivity in multiple crosslinked systems is often difficult to achieve. Here, we show that rapid unambiguous characterisation of disulfide connectivity can be achieved through direct mass spectrometric CID fragmentation of the disulfide intact polypeptides. The method requires a direct mass spectrometric fragmentation of the native disulfide bonded polypeptides and subsequent analysis using a newly developed program, DisConnect. Technical difficulties involving direct fragmentation of proteins are surmounted by an initial proteolytic nick and subsequent determination of the structures of these proteolytic peptides through DisConnect. While the connectivity in proteolytic fragments containing one cystine is evident from the MS profile alone, those with multiple cystines are subjected to subsequent mass spectrometric fragmentation. The wide applicability of this method is illustrated using examples of peptide hormones, peptide toxins, proteins, and disulfide foldamers of a synthetic analogue of a marine peptide toxin. The method, coupled with DisConnect, provides an unambiguous, straightforward approach, especially useful for the rapid screening of the disulfide crosslink fidelity in recombinant proteins, determination of disulfide linkages in natural peptide toxins and characterization of folding intermediates encountered in oxidative folding pathways.

Item Type:	Journal Article
Publication:	Molecular BioSystems
Publisher:	Royal Society of Chemistry
Additional Information:	Copyright of this article belongs to Royal Society of Chemistry.
Department/Centre:	Division of Biological Sciences > Molecular Biophysics Unit
Date Deposited:	18 Jun 2013 11:37
Last Modified:	28 Aug 2014 06:57
URI:	http://eprints.iisc.ac.in/id/eprint/46737

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