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.

Collision-Induced Unfolding of Native-like Protein Ions Within a Trapped Ion Mobility Spectrometry Device.

Native mass spectrometry and collision-induced unfolding (CIU) workflows continue to grow in utilization due to their ability to rapidly characterize protein conformation and stability. To perform these experiments, the instrument must be capable of collisionally activating ions prior to ion mobility spectrometry (IMS) analyses. Trapped ion mobility spectrometry (TIMS) is an ion mobility implementation that has been increasingly adopted due to its inherently high resolution and reduced instrumental footprint. In currently deployed commercial instruments, however, typical modes of collisional activation do not precede IMS analysis, and thus, the instruments are incapable of performing CIU. In this work, we expand on a recently developed method of activating protein ions within the TIMS device and explore its analytical utility toward the unfolding of native-like protein ions. We demonstrate the unfolding of native-like ions of ubiquitin, cytochrome C, ß-lactoglobulin, and carbonic anhydrase. These ions undergo extensive unfolding upon collisional activation. Additionally, the improved resolution provided by the TIMS separation uncovers previously obscured unfolding complexity.