4 research outputs found
Quantifying Protection in Disordered Proteins Using Millisecond Hydrogen Exchange-Mass Spectrometry and Peptic Reference Peptides
The extent and location of transient
structure in intrinsically
disordered proteins (IDPs) provide valuable insights into their conformational
ensembles and can lead to a better understanding of coupled binding
and folding. Millisecond amide hydrogen exchange (HX) can provide
such information, but it is difficult to quantify the degree of transient
structuring. One reason is that transiently disordered proteins undergo
HX at rates only slightly slower than the rate of amide HX by an unstructured
random coil, the chemical HX rate. In this work, we evaluate several
different methods of obtaining an accurate model for the chemical
HX rate suitable for millisecond hydrogen exchange mass spectrometry
(HX-MS) analysis of disordered proteins: (1) calculations using the
method of Englander [Bai, Y., et al. (1993) <i>Proteins</i> <i>17</i>, 75–86], (2) measurement of HX in the
presence of 6 M urea or 3 M guanidinium chloride, and (3) measurement
of HX by peptide fragments derived directly from the proteins of interest.
First, using unstructured model peptides and disordered domains of
the activator for thyroid and retinoid receptors and the CREB binding
protein as the model IDPs, we show that the Englander method has slight
inaccuracies that lead to underestimation of the chemical exchange
rate. Second, HX-MS measurements of model peptides show that HX rates
are changed dramatically by high concentrations of the denaturant.
Third, we find that measurements of HX by reference peptides from
the proteins of interest provide the most accurate approach for quantifying
the extent of transient structure in disordered proteins by millisecond
HX-MS
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