Rapid Online Oxidation of Proteins and Peptides via Electrospray-Accelerated Ozonation

When combined with mass spectrometry, solution-phase labeling such as oxidative foot printing, hydrogen/deuterium exchange, and covalent labeling are powerful tools for the analysis of protein conformation. The throughput of these work-flows, however, is frequently limited due to their reliance on slow labeling reactions, proteolysis, and chromatographic separations to be fully realized. Here, we present an ozone-driven oxidation reaction that occurs on the microsecond time-scale during the electrospray ionization (ESI) process. Selective oxidation of methionine and tryptophan residues in pep-tides and proteins occurs spontaneously upon the introduction of ozone into the ESI chamber. Trp and Met residues are frequently buried in folded proteins and thus, when applied to natively folded cytochrome C and carbonic anhydrase, little oxidation is observed. When these proteins are denatured and ozonated, a dramatic increase in the number of oxidation events and yield is measured. This methodology’s applicability to any instrument equipped with an ESI source, facile interpretation of results, limited sample handling, high-throughput nature, and rapid labeling reaction makes this technique a promising new tool for the analysis of protein folding.