UV Photodissociation Mass Spectrometry Accurately Localize Sites of Backbone Deuteration in Peptides.

Ulrik H Mistarz,Pernille F Jensen,Jeffery M Brown,Perdita E Barran,Kasper D Rand,Bruno Bellina

doi:10.1021/acs.analchem.7b04683

Abstract

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is now a routinely used technique to inform on protein structure, dynamics, and interactions. Localizing the incorporated deuterium content on a single residue basis increases the spatial resolution of this technique enabling detailed structural analysis. Here, we investigate the use of ultraviolet photodissociation (UVPD) at 213 nm to measure deuterium levels at single residue resolution in HDX-MS experiments. Using a selectively labeled peptide, we show that UVPD occurs without H/D scrambling as the peptide probe accurately retains its solution-phase deuterium labeling pattern. Our results indicate that UVPD provides an attractive alternative to electron mediated dissociation for increasing the spatial resolution of the HDX-MS experiment, capable of yielding high fragmentation efficiency, high fragment ion diversity, and low precursor ion charge-state dependency.

Highlights

D etailed insight into the dynamic conformation of proteins is critical for understanding and deciphering the molecular mechanisms of most cellular processes.[1]
Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a routinely used technique to inform on protein structure, dynamics, and interactions
Using a selectively labeled peptide, we show that ultraviolet photodissociation (UVPD) occurs without H/D scrambling as the peptide probe accurately retains its solution-phase deuterium labeling pattern

Summary

Analytical Chemistry

Letter accessible chromophore becomes the amide group (with oribital transitions to π* either from π at 193 nm or from n at 213 nm).[9,21,22] The absorption of a single photon at these wavelengths deposits a high amount of energy (7.9 eV at 157 nm, 6.4 eV at 193 nm, and 5.8 eV at 213 nm) which exceeds peptide bond energies of 3−4 eV21 resulting in comprehensive fragmentation of both peptides and intact proteins, largely independent of ion charge and size.[9]. Letter formed during 213 nm UVPD of peptide P1 precluded reliable quantitative analysis, our results are in line with mechanistic studies of UVPD at 157 nm suggesting that all fragment ions from UVPD using short-wavelength lasers originate from fast radical-based mechanisms.[26] Our findings indicate that UVPD at 213 nm offers a powerful complement to ETD in bottom-up HDX-MS/MS workflows, with the potential to provide alternative fragmentation, with less dependency on size and charge state of the precursor ion,[27] and even allow the extraction of site-specific information for singly charged peptides.[22] In this study, UVPD was performed under settings providing optimal fragment ion yields during a direct infusion experiment enabling quantitation of H/D scrambling at maximum sensitivity.

■ ACKNOWLEDGMENTS

Findings

■ REFERENCES