A mass spectrometric approach to the characterization of protein folding reactions

Abstract

The arsonous acids melarsen oxide and pyridinyl-3-arsonous acid were successfully applied for chemical trapping of otherwise tran sient (un)folding intermediates. The trapped and arsonous acid-modified peptides and proteins were amenable to direct molecular weight determination by mass spectrometry, e.g. by nanoelectrospray ionization mass spectrometry or matrix-assisted laser desorption/ionization mass spectrometry. Arsonous acids exhibited several advantageous features such as (i) cross-linking two closely spaced thiol groups providing detailed tertiary structure information, (ii) high solubility as monomeric and oligomeric deriva tives in aqueous solution, (iii) adding a relatively large mass increment to proteins upon single modification and (iv) performing fast and specific modification of bis-thiol groups in proteins to form stable structures without any side reactions even with a high molar excess of arsonous acids. Bis-cysteine thiol-selective modification of vasopressin with pyridinyl-3-arsonous acid was confirmed by Fourier transform ion cyclotron resonance mass spectrometry measurements. The major product obtained by denaturing unfolding of bovine pancreatic trypsin inhibitor (BPTI) and trapping with melarsen oxide eluted like folded BPTI in high-performance liquid chromatography analyses, indicating a “native-like” structure of this folding intermediate. The results obtained from the investigated model compounds suggest a general applicability of arsonous acids for selective bis-thiol modifications, enabling the study of protein folding reactions by mass spectrometry.