Selective bridging of bis-cysteinyl residues by arsonous acid derivatives as an approach to the characterization of protein tertiary structures and folding pathways by mass spectrometry

Bis-cysteine selective modifications were successfully applied with melarse n oxide (MEL), an arsonous acid derivative, for tertiary structural studies of peptides and a model protein. The arsonous acid modified peptides and p roteins were amenable to direct characterizations by mass spectrometry, e.g ., direct molecular weight determinations and mass spectrometric peptide ma pping that identified stoichiometry and sites of modification, respectively . Proteolytic digestion and mass spectrometric fragmentation of modified ox ytocin showed that MEL-bridged peptide derivatives are structural homologue s to the disulfide-bonded macrocyclic peptides. Mass spectrometric analyses determined the MEL modification site in partially reduced and selectively modified bovine pancreatic trypsin inhibitor (BPTI) bridging Cys-14 and Cys -38. The BPTI MEL derivative was resistant to proteolysis by both Lys-C and trypsin and thus represented a rigid structure like native BPTI. MEL exhib ited several advantageous features such as (i) cross-linking two closely sp aced thiol groups, providing detailed tertiary structure information; (ii) high solubility as monomeric ortho acid in aqueous and organic solutions; ( iii) adding a relatively large mass increment to proteins upon single modif ication; (iv) enabling UV monitoring of the derivatization due to a strong chromophor; and (v) performing fast and specific modifications of bis-thiol groups in proteins to form stable structures without any side reactions ev en with a high molar excess of MEL. The investigated physical and chemical properties of MEL suggest general applicability for selective bis-thiol mod ifications, enabling protein structure-function studies in both soluble and membrane proteins and the study of protein-folding reactions. (C) 1998 Aca demic Press.