Defining the Disulfide Bonds of Insulin-like Growth Factor-binding Protein-5 by Tandem Mass Spectrometry with Electron Transfer Dissociation and Collision-induced Dissociation

Mahta Nili,Aditi Mukherjee,Ujwal Shinde,Larry David,Peter Rotwein

doi:10.1074/jbc.m111.285528

Abstract

The six high-affinity insulin-like growth factor-binding proteins (IGFBPs) comprise a conserved family of secreted molecules that modulate IGF actions by regulating their half-life and access to signaling receptors, and also exert biological effects that are independent of IGF binding. IGFBPs are composed of cysteine-rich amino- (N-) and carboxyl- (C-) terminal domains, along with a cysteine-poor central linker segment. IGFBP-5 is the most conserved IGFBP, and contains 18 cysteines, but only 2 of 9 putative disulfide bonds have been mapped to date. Using a mass spectrometry (MS)-based strategy combining sequential electron transfer dissociation (ETD) and collision-induced dissociation (CID) steps, in which ETD fragmentation preferentially induces cleavage of disulfide bonds, and CID provides exact disulfide linkage assignments between liberated peptides, we now have definitively mapped 5 disulfide bonds in IGFBP-5. In addition, in conjunction with ab initio molecular modeling we are able to assign the other 4 disulfide linkages to within a GCGCCXXC motif that is conserved in five IGFBPs. Because of the nature of ETD fragmentation MS experiments were performed without chemical reduction of IGFBP-5. Our results not only establish a disulfide bond map of IGFBP-5 but also define a general approach that takes advantage of the specificity of ETD and the scalability of tandem MS, and the predictive power of ab initio molecular modeling to characterize unknown disulfide linkages in proteins.

Highlights

2 of 9 putative disulfide bonds have been mapped for insulin-like growth factor-binding proteins (IGFBPs)-5
Amino Acid Substitution Mutations in the N-terminal Domain of IGFBP-5 that Reduce IGF Binding Do Not Alter Disulfide Bonds—Previous NMR studies had mapped the disulfide bond between Cys54 in P4 and Cys80 in P6, using a miniIGFBP-5 protein as the starting material [36]. We analyzed this linkage in full-length IGFBP-5 and compared it to results obtained with a N-terminal domain amino acid substitution mutant involving residues K68N, P69Q, L70Q, L73Q, and L74Q to determine whether disruption of this disulfide bond might account for the diminished IGF binding affinity of the latter protein [35, 37]
In this study we have shown that the 18 cysteines in mouse IGFBP-5 form 9 disulfide bonds

Summary

Background

Results: Using a MS-based strategy combining ETD and CID, and ab initio molecular modeling, we have mapped all 9 disulfide bonds in IGFBP-5. Our results establish a disulfide bond map of IGFBP-5 and define a general approach that takes advantage of the specificity of ETD and the scalability of tandem MS, and the predictive power of ab initio molecular modeling to characterize unknown disulfide linkages in proteins. As CID spectra of peptides containing this cysteine-rich motif could not precisely assign the disulfide linkages within the N-terminal segment of IGFBP-5, we employed unconstrained ab initio modeling to further refine the map. Our results demonstrate the power of a combined approach employing both sequential MS and ab initio molecular modeling to identify and characterize disulfide bonds in a protein, and define a complete disulfide linkage map for IGFBP-5. We find that amino acid substitution mutations in N-terminal domain residues that are critical for maintaining ligand binding affinity (K68, P69, L70, L73, and L74) have a minimal impact on the global tertiary structure of IGFBP-5

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION