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

Mass spectrometry-based analyses of protein conformation continue to grow in utilization due their speed, low sample requirements, and applicability to most protein systems. These techniques typically rely on chemical derivatization of proteins and as with all label-based analyses must ensure the integrity of the protein conformation throughout the duration of the labeling reaction. Hydroxyl radical footprinting of proteins and the recently developed fast fluoroalkylation of proteins attempt to bypass this consideration via rapid reactions that occur on time scales faster than protein folding, but they often require microfluidic setups or electromagnetic radiation sources. In this work, we demonstrate that ozonation of proteins and peptides, which normally occurs in the second to minute time scales, can be accelerated to the submillisecond to millisecond time scale with an electrospray ionization source. This rapid ozonation results in selective labeling of tryptophan and methionine residues. When applied to cytochrome C and carbonic anhydrase, this labeling technique is sensitive to solution conditions and correlates with solution-phase analyses of conformation. While significant work is still needed to characterize this fast chemical labeling strategy, it requires no complicated sample handling, electromagnetic radiation sources, or microfluidic systems outside of the electrospray source and may represent a facile alternative to other rapid labeling technologies that are utilized today.