Hydrogen-deuterium Exchange Labeling Research Articles

Epitope Mapping for a Preclinical Bevacizumab (Avastin) Biosimilar on an Extended Construct of Vascular Endothelial Growth Factor A Using Millisecond Hydrogen-Deuterium Exchange Mass Spectrometry.

The success of bevacizumab (Avastin), a monoclonal antibody (mAb) anticancer drug targeting vascular endothelial growth factor A (VEGF-A), has motivated the development of biosimilars. Establishing target epitope similarity using epitope mapping is a critical step in preclinical mAb biosimilar development. Here we use time-resolved electrospray ionization hydrogen-deuterium exchange (HDX) mass spectrometry to rapidly compare the epitopes of commercial Avastin and a biosimilar in preclinical development (ApoBev) on an extended construct of VEGF-A. The Avastin and ApoBev epitopes determined in our experiments agree with each other and with the known epitope derived from the Avastin Fab domain/truncated VEGF co-crystal structure. However, subtly different allosteric effects observed exclusively at short (millisecond) HDX labeling times may reflect a slightly different binding mode for ApoBev.

Contemporary hydrogen deuterium exchange mass spectrometry.

Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) emerged as a tool for biochemistry and structural biology around 25 years ago. It has since become a key approach for studying protein dynamics, protein-ligand interactions, membrane proteins and intrinsically disordered proteins (IDPs). In HDX labeling, proteins are exposed to deuterated solvent (usually D2O) for a variable 'labeling time', resulting in isotope exchange of unprotected labile protons on the amide backbone and amino acid side chains. By comparing the levels of deuterium uptake in different regions of a protein, information on conformational and dynamic changes in the system can be acquired. When coupled with MS, HDX is suitable for probing allosteric effects in catalysis and ligand binding, epitope mapping, validation of biosimilars, drug candidate screening and mapping membrane-protein interactions among many other bioanalytical applications. This review introduces HDX-MS via a brief description of HDX-MS development, followed by an overview of HDX theory and ultimately an outline of methods and procedures involved in performing HDX-MS experiments.

Characterization of the Retro-[3+2] Reaction of Protonated 2,3′-Bisindolylmethanes by Electrospray Ionization–Tandem Mass Spectrometry

ABSTRACTTwelve 2,3′-bisindolylmethanes with various substituents were investigated using electrospray ionization quadrupole time-of-flight tandem mass spectrometry in positive ion mode. A retro-[3+2] reaction was observed in the collision-induced dissociation spectra of protonated 2,3′-bisindolylmethanes for the first time. The mechanism of retro-[3+2] reaction was concerted or stepwise. For the concerted pathway, carbon–carbon bonds of a protonated compound simultaneously cracked and the m/z 208 ion ([C15H10D2N]+) was observed with hydrogen–deuterium exchange labeling. The stepwise pathway goes through 1,3-hydrogen migration twice and the m/z 208 ion ([C15H10D2N]+) and m/z 207 ion ([C15H11DN]+) were detected with deuterium labeling. In the deuterium-labeled tandem mass spectrum for one compound, only the peak at m/z 208 was present at high abundance, suggesting that the concerted pathway is more likely. In addition, the substituents have no obvious trends on the ratios of the product intensity to the base intensity, further supporting the concerted pathway.

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Characterizing rapid, activity‐linked conformational transitions in proteins via sub‐second hydrogen deuterium exchange mass spectrometry

This review outlines the application of time-resolved electrospray ionization mass spectrometry (TRESI-MS) and hydrogen-deuterium exchange (HDX) to study rapid, activity-linked conformational transitions in proteins. The method is implemented on a microfluidic chip which incorporates all sample-handling steps required for a 'bottom-up' HDX workflow: a capillary mixer for sub-second HDX labeling, a static mixer for HDX quenching, a microreactor for rapid protein digestion, and on-chip electrospray. By combining short HDX labeling pulses with rapid digestion, this approach provides a detailed characterization of the structural transitions that occur during protein folding, ligand binding, post-translational modification and catalytic turnover in enzymes. This broad spectrum of applications in areas largely inaccessible to conventional techniques means that microfluidics-enabled TRESI-MS/HDX is a unique and powerful approach for investigating the dynamic basis of protein function.

Loading, stationary phase, and salt effects during hydrophobic interaction chromatography: α-Lactalbumin is stabilized at high loadings

Amide hydrogen-deuterium exchange labeling has been used to study the effects of salt and protein loading on α-lactalbumin (BLA) stability during hydrophobic interaction chromatography (HIC). Stability in the adsorbed phase increased dramatically with increasing loading, and unfolding was nearly undetectable close to the resin saturation capacity. We also found that a butyl surface destabilized BLA more than a phenyl surface, despite the fact that BLA was bound more strongly on the phenyl surface. These observations have important implications for HIC process design and indicate that in some cases column capacity does not have to be sacrificed to preserve protein stability.

Protein Folding Mechanisms Studied by Time-Resolved Electrospray Mass Spectrometry

Mass spectrometry (MS) owes its remarkable rise as a bioanalytical tool to the development of two “soft” ionization techniques: matrix-assisted laser desorption/ ionization (MALDI) (1,2) and electrospray ionization (ESI) (3). The salient feature of soft ionization is that a wide range of analytes, from small metabolites to very large biomolecular systems, can be transferred intact into the gas phase, thus making them amenable to mass spectrometric analysis. For MALDI-MS, the sample is embedded into a crystalline matrix prior to analysis. For ESI, in contrast, ionization is initiated directly from the liquid phase by spraying analyte solution from the tip of an electrically charged capillary. The small droplets formed by the ESI emitter undergo rapid evaporation and fi ssion, ultimately releasing the analyte as multiply protonated (or deprotonated) species (4).

Structural features of interferon-gamma aggregation revealed by hydrogen exchange.

Using hydrogen-deuterium exchange (HX) and electrospray ionization mass spectrometry, we have investigated the stability and structural changes of recombinant human interferon-gamma (IFN-gamma) during aggregation induced by guanidine hydrochloride (GdnHCl) and potassium thiocyanate. First, HX labeling was initiated after the amorphous aggregates were formed to probe the tertiary structure of the aggregated state. Second, labeling was performed at low protein concentrations to assess stability under aggregation prone conditions. In 1 M GdnHCl, the stability of IFN-gamma was greatly reduced and much less protection from HX in solution was observed. Exchange under these conditions was slower in helix C than in the rest of the protein. Aggregates formed in 1 M GdnHCl showed a HX pattern consistent with a partially unfolded state with an intact helix C. Although aggregates formed in 0.3 M KSCN exhibited a HX pattern similar to those formed in GdnHCl, the solution phase HX pattern in 0.3 M KSCN was surprisingly comparable to that of the native state. Varying the aggregation time before performing HX revealed that KSCN first precipitated native protein and then facilitated partial unfolding of the precipitated protein. These results show that helix C, which forms the hydrophobic core of the IFN-gamma dimer, is highly protected from HX under native conditions, is more stable in GdnHCl than the rest of the protein and remains intact in both GdnHCl- and KSCN-induced aggregates. This suggests that native-state HX patterns may presage regions of the protein susceptible to unfolding during aggregation.

Protein unfolding during reversed-phase chromatography: I. Effect of surface properties and duration of adsorption.

Residue-level features of bovine pancreatic trypsin inhibitor (BPTI) unfolding on reversed-phase chromatography (RPC) surfaces were investigated using hydrogen-deuterium exchange labeling and NMR. A set of silica-based RPC surfaces was used to examine the influence of alkyl chain length and media pore size on adsorbed BPTI conformation. In all cases there was substantial unfolding in the adsorbed state; however, residual protection from exchange was consistently observed. Particle pore size did not influence conformation substantially for C4-alkyl modified silica; however, 120 A pore C18 media produced more hydrogen exchange than any other surface examined. In this case, the radius of curvature inside the pore approaches the size of the BPTI molecule. Generally, the pattern of hydrogen exchange protection was uniform; however, the beta-sheet region was selectively protected on the large-pore C18 media. The beta-sheet region forms a hydrophobic core that forms early when BPTI folds in solution. This suggests that partially unfolded states possessing a native-like structure play an important role in adsorption and elution in RPC. Finally, increased contact time with the surface before elution fostered unfolding and altered chromatographic behavior considerably.

Identification of Protein Adsorbing Site onto Solid Surface Using Hydrogen-Deuterium Exchange Labeling.

Identification of Protein Adsorbing Site onto Solid Surface Using Hydrogen-Deuterium Exchange Labeling.

Studies of protein-protein association between yeast cytochrome c peroxidase and yeast iso-1 ferricytochrome c by hydrogen-deuterium exchange labeling and proton NMR spectroscopy.

Hydrogen-deuterium (H-D) exchange labeling and proton NMR have been applied to study the protein-protein association between cytochrome c peroxidase (CcP) and yeast iso-1 ferricytochrome c. Specifically, the exchange behavior of individual backbone amide protons of yeast iso-1 ferricytochrome c in both CcP-bound (i.e., complexed) and free (i.e., never in the complex) forms has been investigated and used in an attempt to map the binding site of CcP on yeast iso-1 ferricytochrome c when the noncovalent complex was formed in very low salt solution. The exchange rates of certain amino acid amide protons were significantly slowed down, by up to 40-fold, in the complex compared to the free form. The protected regions on iso-1 ferricytochrome c include parts of the 10's helix and the 70's helix surrounding the cytochrome c heme solvent-exposed edge (the so-called "front side" of iso-1 cytochrome c). These regions are very similar to the cytochrome c peroxidase binding interface on iso-1 ferricytochrome c that has been defined by X-ray crystallographic data. This further supports the direct involvement of the front side of iso-1 cytochrome c in binding with cytochrome c peroxidase. The results from our H-D exchange experiments also indicated that the amide proton exchange rates of Trp59, Asp60, and part of the 90's helix, all of which are located on the opposite side (the "back" side) of ferricytochrome c from the heme solvent-exposed edge, are also retarded upon complex formation.

Early hydrogen-bonding events in the folding reaction of ubiquitin.

The formation of hydrogen-bonded structure in the folding reaction of ubiquitin, a small cytoplasmic protein with an extended beta-sheet and an alpha-helix surrounding a pronounced hydrophobic core, has been investigated by hydrogen-deuterium exchange labeling in conjunction with rapid mixing methods and two-dimensional NMR analysis. The time course of protection from exchange has been measured for 26 back-bone amide protons that form stable hydrogen bonds upon refolding and exchange slowly under native conditions. Amide protons in the beta-sheet and the alpha-helix, as well as protons involved in hydrogen bonds at the helix/sheet interface, become 80% protected in an initial 8-ms folding phase, indicating that the two elements of secondary structure form and associate in a common cooperative folding event. Somewhat slower protection rates for residues 59, 61, and 69 provide evidence for the subsequent stabilization of a surface loop. Most probes also exhibit two minor phases with time constants of about 100 ms and 10 s. Only two of the observed residues, Gln-41 and Arg-42, display significant slow folding phases, with amplitudes of 37% and 22%, respectively, which can be attributed to native-like folding intermediates containing cis peptide bonds for Pro-37 and/or Pro-38. Compared with other proteins studied by pulse labeling, including cytochrome c, ribonuclease, and barnase, the initial formation of hydrogen-bonded structure in ubiquitin occurs at a more rapid rate and slow-folding species are less prominent.

Deuterated peptides: Platinum catalyzed exchange labeling of leucine enkephalin and related peptides

Platinum catalyzed hydrogen-deuterium exchange labeling of phenylalanine and tyrosine containing peptides in deuterium oxide is exemplified by the preparation of deuterium labeled leucine enkephalin and related peptides. NMR and mass spectral evidence indicates that the side chains of the aromatic residues are the sites of label incorporation. Time course studies with N-acetyltyrosine and tyrosyl-glycine suggest that the exchange of the aromatic protons occurs most rapidly followed by exchange of the benzylic protons adjacent to the aromatic ring.