Mapping disulfide bonds in insulin with the route 66 method: Selective cleavage of S-C bonds using alkali and alkaline earth metal enolate complexes


Simple and fast identification of disulfide linkages in insulin is demonstrated with a peptic digest using the Route 66 method. This is accomplished by collisional activation of singly and doubly charged cationic Na+ and Ca2+ complexes generated using electrospray ionization mass spectrometry (ESI-MS). Collisional activation of doubly charged metal complexes of peptides with intermolecular disulfide linkages yields two sets of singly charged paired products separated by 66 mass units resulting from selective S-C bond cleavages. Highly selective elimination of 66 mass units, which corresponds to the molecular weight of hydrogen disulfide (H2S2), is observed from singly charged metal complexes of peptides with disulfide linkages. The mechanism proposed for these processes is initiated by formation of a metal-stabilized enolate at Cys, followed by cleavage of the S-C bond. Further activation of the products yields sequence information that facilitates locating the position of the disulfide linkages in the peptic digest fragments. For example, the doubly charged Ca2+ complex of the peptic digest product GIVEQCCASVCSL/FVNQHLCGSHL yields paired products separated by 66 mass units resulting from selective S-C bond cleavages at an intermolecular disulfide linkage under low-energy collision-induced dissociation. Further activation of the product comprising the A chain reveals the presence of a second disulfide bridge, an intramolecular linkage. Experimental and theoretical studies of the disulfide linked model peptides provide mechanistic details for the selective cleavage of the S-C bond.


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Correspondence to Hugh I. Kim or J. L. Beauchamp.

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Published online October 11, 2008

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Kim, H.I., Beauchamp, J.L. Mapping disulfide bonds in insulin with the route 66 method: Selective cleavage of S-C bonds using alkali and alkaline earth metal enolate complexes. J Am Soc Mass Spectrom 20, 157–166 (2009).

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  • Disulfide Bond
  • Collisional Activation
  • Disulfide Linkage
  • Intermolecular Disulfide Bond
  • Dehydroalanine