9 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 fifteen 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 (1σx ̅ ), and all laboratories achieved 〈 sLab 〉 ≤ 0.4 Da. For immersions of protein at THDX = (3.6 to 25) oC 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 Labs ( tHDX ) = (9.0 ± 0.9) % (1σ). A 9 laboratory cohort that immersed samples at THDX = 25 oC exhibited reproducibility of σreproducibility25C cohort ( tHDX ) = (6.5 ± 0.6) % for back-exchange corrected, deuterium uptake measurements
Probing adenylation: using a fluorescently labelled ATP probe to directly label and immunoprecipitate VopS substrates.
The bacterial effector VopS from Vibrio parahaemolyticus modifies host Rho GTPases to prevent downstream signalling, which leads to cell rounding and eventually apoptosis. While previous studies have used [alpha-(32)P] ATP for studying this enzyme, we sought to develop a non-radioactive chemical probe of VopS function. To guide these studies, the kinetic parameters were determined for a variety of nucleotides and the results indicated that the C6 position of adenosine was amenable to modification. Since Fl-ATP is a commercially available ATP analogue that is fluorescently tagged at the C6 position, we tested it as a VopS substrate, and the results show that VopS uses Fl-ATP to label Cdc42 in vitro and in MCF7 whole cell extracts. The utility of this probe was further demonstrated by immunoprecipitating Fl-ATP labeled Cdc42 as well as several novel substrate proteins. The proteins, which were identified by LC-MS/MS, include the small GTPases Rac1 and Cdc42 as well as several proteins that are potential VopS substrates and may be important for V. parahaemolyticus pathology. In total, these studies identify Fl-ATP as a valuable chemical probe of protein AMPylation
Estimation of Hydrogen-Exchange Protection Factors from MD Simulation Based on Amide Hydrogen Bonding Analysis
Hydrogen
exchange (HX) studies have provided critical insight into our understanding
of protein folding, structure, and dynamics. More recently, hydrogen
exchange mass spectrometry (HX-MS) has become a widely applicable
tool for HX studies. The interpretation of the wealth of data generated
by HX-MS experiments as well as other HX methods would greatly benefit
from the availability of exchange predictions derived from structures
or models for comparison with experiment. Most reported computational
HX modeling studies have employed solvent-accessible-surface-area
based metrics in attempts to interpret HX data on the basis of structures
or models. In this study, a computational HX-MS prediction method
based on classification of the amide hydrogen bonding modes mimicking
the local unfolding model is demonstrated. Analysis of the NH bonding
configurations from molecular dynamics (MD) simulation snapshots is
used to determine partitioning over bonded and nonbonded NH states
and is directly mapped into a protection factor (PF) using a logistics
growth function. Predicted PFs are then used for calculating deuteration
values of peptides and compared with experimental data. Hydrogen exchange
MS data for fatty acid synthase thioesterase (FAS-TE) collected for
a range of pHs and temperatures was used for detailed evaluation of
the approach. High correlation between prediction and experiment for
observable fragment peptides is observed in the FAS-TE and additional
benchmarking systems that included various apo/holo proteins for which
literature data were available. In addition, it is shown that HX modeling
can improve experimental resolution through decomposition of in-exchange
curves into rate classes, which correlate with prediction from MD.
Successful rate class decompositions provide further evidence that
the presented approach captures the underlying physical processes
correctly at the single residue level. This assessment is further
strengthened in a comparison of residue resolved protection factor
predictions for staphylococcal nuclease with NMR data, which was also
used to compare prediction performance with other algorithms described
in the literature. The demonstrated transferable and scalable MD based
HX prediction approach adds significantly to the available tools for
HX-MS data interpretation based on available structures and models
Estimation of Hydrogen-Exchange Protection Factors from MD Simulation Based on Amide Hydrogen Bonding Analysis
Hydrogen
exchange (HX) studies have provided critical insight into our understanding
of protein folding, structure, and dynamics. More recently, hydrogen
exchange mass spectrometry (HX-MS) has become a widely applicable
tool for HX studies. The interpretation of the wealth of data generated
by HX-MS experiments as well as other HX methods would greatly benefit
from the availability of exchange predictions derived from structures
or models for comparison with experiment. Most reported computational
HX modeling studies have employed solvent-accessible-surface-area
based metrics in attempts to interpret HX data on the basis of structures
or models. In this study, a computational HX-MS prediction method
based on classification of the amide hydrogen bonding modes mimicking
the local unfolding model is demonstrated. Analysis of the NH bonding
configurations from molecular dynamics (MD) simulation snapshots is
used to determine partitioning over bonded and nonbonded NH states
and is directly mapped into a protection factor (PF) using a logistics
growth function. Predicted PFs are then used for calculating deuteration
values of peptides and compared with experimental data. Hydrogen exchange
MS data for fatty acid synthase thioesterase (FAS-TE) collected for
a range of pHs and temperatures was used for detailed evaluation of
the approach. High correlation between prediction and experiment for
observable fragment peptides is observed in the FAS-TE and additional
benchmarking systems that included various apo/holo proteins for which
literature data were available. In addition, it is shown that HX modeling
can improve experimental resolution through decomposition of in-exchange
curves into rate classes, which correlate with prediction from MD.
Successful rate class decompositions provide further evidence that
the presented approach captures the underlying physical processes
correctly at the single residue level. This assessment is further
strengthened in a comparison of residue resolved protection factor
predictions for staphylococcal nuclease with NMR data, which was also
used to compare prediction performance with other algorithms described
in the literature. The demonstrated transferable and scalable MD based
HX prediction approach adds significantly to the available tools for
HX-MS data interpretation based on available structures and models
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