13 research outputs found
Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments.
Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances
Interlaboratory Comparison of Hydrogen-Deuterium Exchange Mass Spectrometry Measurements of the Fab fragment of NISTmAb
Hydrogen–deuterium
exchange mass spectrometry (HDX-MS) is an established, powerful tool
for investigating protein–ligand interactions, protein folding,
and protein dynamics. However, HDX-MS is still an emergent tool for
quality control of biopharmaceuticals and for establishing dynamic
similarity between a biosimilar and an innovator therapeutic. Because
industry will conduct quality control and similarity measurements
over a product lifetime and in multiple locations, an understanding
of HDX-MS reproducibility is critical. To determine the reproducibility
of continuous-labeling, bottom-up HDX-MS measurements, the present
interlaboratory comparison project evaluated deuterium uptake data
from the Fab fragment of NISTmAb reference material (PDB: 5K8A) from 15 laboratories.
Laboratories reported ∼89 800 centroid measurements
for 430 proteolytic peptide sequences of the Fab fragment (∼78 900
centroids), giving ∼100% coverage, and ∼10 900
centroid measurements for 77 peptide sequences of the Fc fragment.
Nearly half of peptide sequences are unique to the reporting laboratory,
and only two sequences are reported by all laboratories. The majority
of the laboratories (87%) exhibited centroid mass laboratory repeatability
precisions of ⟨sLab⟩ ≤
(0.15 ± 0.01) Da (1σx̅). All laboratories
achieved ⟨sLab⟩ ≤ 0.4 Da. For immersions
of protein at THDX = (3.6 to 25) °C
and for D2O exchange times of tHDX = (30 s to 4 h) the reproducibility of back-exchange corrected,
deuterium uptake measurements for the 15 laboratories is σreproducibility15 Laboratories(tHDX) = (9.0 ± 0.9) % (1σ).
A nine laboratory cohort that immersed samples at THDX = 25 °C exhibited reproducibility of σreproducibility25C cohort(tHDX) = (6.5 ± 0.6) % for back-exchange
corrected, deuterium uptake measurements