Mapping disulfide bonds in insulin with the route 66 method: selective cleavage of SOC bonds using alkali and alkaline earth metal enolate complexes

Abstract

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 Ca^2+ 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 (H_2S_2), 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 Ca^(2+) 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.