TY - JOUR
T1 - Mapping Disulfide Bonds in Insulin with the Route 66 Method
T2 - Selective Cleavage of S{single bond}C Bonds Using Alkali and Alkaline Earth Metal Enolate Complexes
AU - Kim, Hugh I.
AU - Beauchamp, J. L.
N1 - Funding Information:
The research described herein was carried out at the Beckman Institute and the Noyes Laboratory of Chemical Physics at the California Institute of Technology. We appreciate the support provided by the Beckman Institute Mass Spectrometry Resource Center and the Planetary Science section, Jet Propulsion Laboratory, California Institute of Technology. Partial support was also provided by the National Science Foundation under Grant CHE-0416381.
PY - 2009/1
Y1 - 2009/1
N2 - 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{single bond}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{single bond}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{single bond}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{single bond}C bond.
AB - 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{single bond}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{single bond}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{single bond}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{single bond}C bond.
UR - http://www.scopus.com/inward/record.url?scp=57649156395&partnerID=8YFLogxK
U2 - 10.1016/j.jasms.2008.10.003
DO - 10.1016/j.jasms.2008.10.003
M3 - Article
C2 - 18990587
AN - SCOPUS:57649156395
SN - 1044-0305
VL - 20
SP - 157
EP - 166
JO - Journal of the American Society for Mass Spectrometry
JF - Journal of the American Society for Mass Spectrometry
IS - 1
ER -