3 research outputs found
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
Comparing Hydrogen Deuterium Exchange and Fast Photochemical Oxidation of Proteins: a Structural Characterisation of Wild-Type and ΔN6 β₂-Microglobulin
Hydrogen deuterium exchange (HDX) coupled to mass spectrometry (MS) is a well-established technique employed in the field of structural MS to probe the solvent accessibility, dynamics and hydrogen bonding of backbone amides in proteins. By contrast, fast photochemical oxidation of proteins (FPOP) uses hydroxyl radicals, liberated from the photolysis of hydrogen peroxide, to covalently label solvent accessible amino acid side chains on the microsecond-millisecond timescale. Here, we use these two techniques to study the structural and dynamical differences between the protein β₂-microglobulin (β₂m) and its amyloidogenic truncation variant, ΔN6. We show that HDX and FPOP highlight structural/dynamical differences in regions of the proteins, localised to the region surrounding the N-terminal truncation. Further, we demonstrate that, with carefully optimised LC-MS conditions, FPOP data can probe solvent accessibility at the sub-amino acid level, and that these data can be interpreted meaningfully to gain more detailed understanding of the local environment and orientation of the side chains in protein structures