title: Rapid mass spectrometric determination of disulfide connectivity in peptides and proteins.
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.
authors: Molecular Biophysics Unit, Indian Institute of Science, Bangalore-560012, India.
authors: Moitrayee
authors: M
authors: Bhattacharyya
authors: Kallol
authors: K
authors: Gupta
authors: Konkallu Hanumae
authors: KH
authors: Gowd
authors: Padmanabhan
authors: P
authors: Balaram
mesh_terms: D003553:Cystine / Q000737:chemistry; D004220:Disulfides / Q000032:analysis / Q000737:chemistry*; D007768:Lactalbumin / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D045265:Natriuretic Peptides / Q000737:chemistry / Q000378:metabolism; D009498:Neurotoxins / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D004220:Disulfides; D045265:Natriuretic Peptides; D009498:Neurotoxins; D010446:Peptide Fragments; D011506:Proteins; D011994:Recombinant Proteins; C084362:uroguanylin; D003553:Cystine; D007768:Lactalbumin