This project applies hydrogen/deuterium exchange mass spectrometry (HDX-MS) for determining the dynamical structure of therapeutic antibodies, glycoproteins, and proteins; transmembrane protein receptors; and ligand-receptor interactions. Topics of particular interest include protein-ligand interactions, structure-function relationships between glycan structure and glycoprotein folding, and comparability measurements among innovator drugs and biosimilar drug candidates. This project aims to validate HDX-MS measurement technology by conducting interlaboratory comparisons and to improve measurement accuracy through parallel HDX-MS (including analyses that employ electron transfer dissociation) and HDX-NMR studies of selected proteins and glycoproteins.
This project is designed to improve, test, and validate hydrogen/deuterium exchange mass spectrometry (HDX-MS) metrology for the determination of dynamical properties of therapeutic proteins and glycoproteins.
The research project uses HDX-MS to measure the D-for-H exchange rates of the amide groups along the backbone of a protein in D2O solution. These rates indicate the protection factors of the amide groups. The protection factors are characteristic of higher order structural features of proteins (e.g., α-helixes, β-sheets ...) that are stabilized through hydrogen bonding, disulfide bonds, electrostatic interactions, and hydrophobic forces. Since protection factors can change as the protein binds with ligands or undergoes folding, HDX-MS can provide sensitive diagnostic data evidencing structural differences. HDX-MS is a rapidly evolving metrology. Numerous improvements in automation, instrument resolution and accuracy, and new, more powerful software are being introduced.
An interlaboratory study involving 37 co-authors in 15 unharmonized laboratories determined the reproducibility of continuous-labeling, bottom-up HDX-MS measurements by measuring the deuterium uptake of Fab of NISTmAb (PDB: 5K8A) for imersions in D2O for tHDX = (30 to 14400) s and THDX = (25, 21, 3.6) oC. Each laboratory was sent a standardized kit containing Fab protein the reagents necessary to conduct experiments.
Laboratories reported ~ 89,800 centroid measurements for 430 proteolytic peptide sequences of the Fab fragment (~78,900 centroids), giving nearly 100 % coverage, and approximately 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 ≤ (0.15 ±0.01) Da (1σx). All laboratories achieved ≤ 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 (9.0 ±0.9) % (1s). A 9 laboratory cohort that immersed samples at THDX = 25 oC exhibited reproducibility of (6.5 ±0.6) % for back-exchange corrected, deuterium uptake measurements. (See refs 1, 2 below.)
NIST researchers have developed a new, automated approach to study the structural dynamics of membrane proteins, the most common targets for pharmaceuticals. The new method involves isolation of proteins from their phospholipid bilayer using a scheme that is compatible with robotic handling. The key development was the introduction of ZrO2 microbeads, which strongly coordinate with the membrane phospholipids, and then removal of microbead-coordinated phospholipids by inline nanofilters. The protein is thus freed from phospholipids, which interfere with analysis by LC-MS.
Previous HDX-MS publications on membrane protein dynamics removed phospholipids manually, requiring arduous, hours-long laboratory work. The new approach was demonstrated on the transmembrane protein FcγRIIa, and showed information about the regions outside of, across, and inside of its liposomal membrane, enabling measurement of structural dynamics of the whole protein. This approach is the first to enable fully automated HDX-MS on full-length transmembrane proteins in lipid bilayers. (See ref. 3 below.)
Demonstrated that HDX-MS can detect dynamical differences among glycoforms of the same glycoprotein. (Ref. 8)
Developed a method that measures D-uptake by glycans within glycoproteins (Ref. 8).
Used HDX-MS and mutagenesis to find the epitope of Proliferating Cell Nuclear Antigen (PCNA) and a previously unknown ligand, TIP. This ligand offers a new vector for attacking solid tumor cancers (Ref. 9).