title: Defining the disulfide bonds of insulin-like growth factor-binding protein-5 by tandem mass spectrometry with electron transfer dissociation and collision-induced dissociation.
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.
authors: Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, Oregon 97239-3098, USA.
authors: Mahta
authors: M
authors: Nili
authors: Aditi
authors: A
authors: Mukherjee
authors: Ujwal
authors: U
authors: Shinde
authors: Larry
authors: L
authors: David
authors: Peter
authors: P
authors: Rotwein
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D002460:Cell Line; D003545:Cysteine; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000737:chemistry* / Q000235:genetics; D013058:Mass Spectrometry; D051379:Mice; D008958:Models, Molecular; D010449:Peptide Mapping / Q000379:methods*
chemical_list: D004220:Disulfides; D018975:Insulin-Like Growth Factor Binding Protein 5; D003545:Cysteine