A mass spectrometric approach to the characterization of protein folding reactions

The arsonous acids melarsen oxide and pyridinyl-3-arsonous acid were succes sfully applied for chemical trapping of otherwise transient (un)folding int ermediates. The trapped and arsonous acid-modified peptides and proteins we re amenable to direct molecular weight determination by mass spectrometry, e.g. by nanoelectrospray ionization mass spectrometry or matrix-assisted la ser desorption/ionization mass spectrometry. Arsonous acids exhibited sever al advantageous features such as (i) cross-linking two closely spaced thiol groups providing detailed tertiary structure information, (ii) high solubi lity as monomeric and oligomeric derivatives in aqueous solution, (iii) add ing a relatively large mass increment to proteins upon single modification and (iv) performing fast and specific modification of bis-thiol groups in p roteins to form stable structures without any side reactions even,vith a hi gh molar excess of arsonous acids. Bis-cysteine thiol-selective modificatio n of vasopressin with pyridinyl-3-arsonous acid was confirmed by Fourier tr ansform ion cyclotron resonance mass spectrometry measurements. The major p roduct obtained by denaturing unfolding of bovine pancreatic trypsin inhibi tor (BPTI) and trapping with melarsen oxide eluted like folded BPTI in high -performance liquid chromatography analyses, indicating a "native-like" str ucture of this folding intermediate. The results obtained from the investig ated model compounds suggest a general applicability of arsonous acids for selective bis-thiol modifications, enabling the study of protein folding re actions by mass spectrometry.