HDX-MS Dotaz Zobrazit nápovědu
Protein glycosylation is one of the most common PTMs and many cell surface receptors, extracellular proteins, and biopharmaceuticals are glycosylated. However, HDX-MS analysis of such important glycoproteins has so far been limited by difficulties in determining the HDX of the protein segments that contain glycans. We have developed a column containing immobilized PNGase Rc (from Rudaea cellulosilytica) that can readily be implemented into a conventional HDX-MS setup to allow improved analysis of glycoproteins. We show that HDX-MS with the PNGase Rc column enables efficient online removal of N-linked glycans and the determination of the HDX of glycosylated regions in several complex glycoproteins. Additionally, we use the PNGase Rc column to perform a comprehensive HDX-MS mapping of the binding epitope of a mAb to c-Met, a complex glycoprotein drug target. Importantly, the column retains high activity in the presence of common quench-buffer additives like TCEP and urea and performed consistent across 114 days of extensive use. Overall, our work shows that HDX-MS with the integrated PNGase Rc column can enable fast and efficient online deglycosylation at harsh quench conditions to provide comprehensive analysis of complex glycoproteins.
- MeSH
- glykopeptidasa MeSH
- glykoproteiny * analýza MeSH
- glykosylace MeSH
- polysacharidy * metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Hydrogen/deuterium exchange monitored by mass spectrometry is a promising technique for rapidly fingerprinting structural and dynamical properties of proteins. The time-dependent change in the mass of any fragment of the polypeptide chain depends uniquely on the rate of exchange of its amide hydrogens, but determining the latter from the former is generally not possible. Here, we show that, if time-resolved measurements are available for a number of overlapping peptides that cover the whole sequence, rate constants for each amide hydrogen exchange (or equivalently, their protection factors) may be extracted and the uniqueness of the solutions obtained depending on the degree of peptide overlap. However, in most cases, the solution is not unique, and multiple alternatives must be considered. We provide a statistical method that clusters the solutions to further reduce their number. Such analysis always provides meaningful constraints on protection factors and can be used in situations in which obtaining more refined experimental data is impractical. It also provides a systematic way to improve data collection strategies to obtain unambiguous information at single-residue level (e.g., for assessing protein structure predictions at atomistic level).
Hydrogen/deuterium exchange (HDX) followed by mass spectrometry detection (MS) provides a fast, reliable, and detailed solution for the assessment of a protein structure. It has been widely recognized as an indispensable tool and already approved by several regulatory agencies as a structural technique for the validation of protein biopharmaceuticals, including antibody-based drugs. Antibodies are of a key importance in life and medical sciences but considered to be challenging analytical targets because of their compact structure stabilized by disulfide bonds and due to the presence of glycosylation. Despite these difficulties, there are already numerous excellent studies describing MS-based antibody structure characterization. In this chapter, we describe a universal HDX-MS workflow. Deeper attention is paid to sample handling, optimization procedures, and feasibility stages, as these elements of the HDX experiment are crucial for obtaining reliable detailed and spatially well-resolved information.
Intrinsic protein dynamics contribute to their biological functions. Rational engineering of protein dynamics is extremely challenging with only a handful of successful examples. Hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) represents a powerful technique for quantitative analysis of protein dynamics. Here we provide a detailed description of the preparation of protein samples, collection of high-quality data, and their in-depth analysis using various computational tools. We illustrate the application of HDX-MS for the study of protein dynamics in the rational engineering of flexible loops in the reconstructed ancestor of haloalkane dehalogenase and Renilla luciferase. These experiments provided unique and valuable data rigorously describing the modification of protein dynamics upon grafting of the loop-helix element. Tips and tricks are provided to stimulate the wider use of HDX-MS to study and engineer protein dynamics.
- Publikační typ
- abstrakt z konference MeSH
The conserved Tweety homolog (TTYH) family consists of three paralogs in vertebrates, displaying a ubiquitous expression pattern. Although considered as ion channels for almost two decades, recent structural and functional analyses refuted this role. Intriguingly, while all paralogs shared a dimeric stoichiometry following detergent solubilization, their structures revealed divergence in their relative subunit orientation. Here, we determined the stoichiometry of intact mouse TTYH (mTTYH) complexes in cells. Using cross-linking and single-molecule fluorescence microscopy, we demonstrate that mTTYH1 and mTTYH3 form tetramers at the plasma membrane, stabilized by interactions between their extracellular domains. Using blue-native PAGE, fluorescence-detection size-exclusion chromatography, and hydrogen/deuterium exchange mass spectrometry (HDX-MS), we reveal that detergent solubilization results in tetramers destabilization, leading to their dissolution into dimers. Moreover, HDX-MS demonstrates that the extracellular domains are stabilized in the context of the tetrameric mTTYH complex. Together, our results expose the innate tetrameric organization of TTYH complexes at the cell membrane. Future structural analyses of these assemblies in native membranes are required to illuminate their long-sought cellular function.
- MeSH
- buněčná membrána MeSH
- detergenty * MeSH
- myši MeSH
- vodík/deuteriová výměna a hmotnostní spektrometrie * MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Identification of a protein minimal fragment amenable to crystallisation can be time- and labour intensive especially if large amounts are required and the protein has a complex fold and functionally important post-translational modifications. In addition, a lack of homologues and structural information can further complicate the design of a minimal expression construct. Recombinant expression in E. coli promises high yields, low costs and fast turnover times, but falls short for many extracellular, eukaryotic proteins. Eukaryotic expression systems provide an alternative but are costly, slow and require special handling and equipment. Using a member of a structurally uncharacterized, eukaryotic receptor family as an example we employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) guided construct design in conjunction with truncation scanning and targeted expression host switching to identify a minimal expression construct that can be produced with high yields and moderate costs.
A comparative canine-human therapeutics model is being developed in B-cell lymphoma through the generation of a hybridoma cell that produces a murine monoclonal antibody specific for canine CD20. The hybridoma cell produces two light chains, light chain-3, and light chain-7. However, the contribution of either light chain to the authentic full-length hybridoma derived IgG is undefined. Mass spectrometry was used to identify only one of the two light chains, light chain-7, as predominating in the full-length IgG. Gene synthesis created a recombinant murine-canine chimeric monoclonal antibody expressing light chain-7 that reconstituted the IgG binding to CD20. Using light chain-7 as a reference sequence, hydrogen deuterium exchange mass spectrometry was used to identify the dominant CDR region implicated in CD20 antigen binding. Early in the deuteration reaction, the CD20 antigen suppressed deuteration at CDR3 (VH). In later time points, deuterium suppression occurred at CDR2 (VH) and CDR2 (VL), with the maintenance of the CDR3 (VH) interaction. These data suggest that CDR3 (VH) functions as the dominant antigen docking motif and that antibody aggregation is induced at later time points after antigen binding. These approaches define a methodology for fine mapping of CDR contacts using nested enzymatic reactions and hydrogen deuterium exchange mass spectrometry. These data support the further development of an engineered, synthetic canine-murine monoclonal antibody, focused on CDR3 (VH), for use as a canine lymphoma therapeutic that mimics the human-murine chimeric anti-CD20 antibody Rituximab.
- MeSH
- antigeny CD20 imunologie MeSH
- chromatografie kapalinová MeSH
- imunoglobulin G chemie MeSH
- kinetika MeSH
- lehké řetězce imunoglobulinů genetika metabolismus MeSH
- lidé MeSH
- monoklonální protilátky chemie genetika MeSH
- nádorové buněčné linie MeSH
- peptidová knihovna MeSH
- psi MeSH
- rekombinantní fúzní proteiny MeSH
- sekvence aminokyselin MeSH
- tandemová hmotnostní spektrometrie MeSH
- těžké řetězce imunoglobulinů genetika metabolismus MeSH
- vazebná místa protilátek MeSH
- vodík/deuteriová výměna a hmotnostní spektrometrie * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- psi MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Hydrogen/deuterium exchange (HDX) is a well-established analytical technique that enables monitoring of protein dynamics and interactions by probing the isotope exchange of backbone amides. It has virtually no limitations in terms of protein size, flexibility, or reaction conditions and can thus be performed in solution at different pH values and temperatures under controlled redox conditions. Thanks to its coupling with mass spectrometry (MS), it is also straightforward to perform and has relatively high throughput, making it an excellent complement to the high-resolution methods of structural biology. Given the recent expansion of artificial intelligence-aided protein structure modeling, there is considerable demand for techniques allowing fast and unambiguous validation of in silico predictions; HDX-MS is well-placed to meet this demand. Here we present a protocol for HDX-MS and illustrate its use in characterizing the dynamics and structural changes of a dimeric heme-containing oxygen sensor protein as it responds to changes in its coordination and redox state. This allowed us to propose a mechanism by which the signal (oxygen binding to the heme iron in the sensing domain) is transduced to the protein's functional domain.
In analogy with many other proteins, Na(+)/Ca(2+) exchangers (NCX) adapt an inverted twofold symmetry of repeated structural elements, while exhibiting a functional asymmetry by stabilizing an outward-facing conformation. Here, structure-based mutant analyses of the Methanococcus jannaschii Na(+)/Ca(2+) exchanger (NCX_Mj) were performed in conjunction with HDX-MS (hydrogen/deuterium exchange mass spectrometry) to identify the structure-dynamic determinants of functional asymmetry. HDX-MS identified hallmark differences in backbone dynamics at ion-coordinating residues of apo-NCX_Mj, whereas Na(+)or Ca(2+) binding to the respective sites induced relatively small, but specific, changes in backbone dynamics. Mutant analysis identified ion-coordinating residues affecting the catalytic capacity (kcat/Km), but not the stability of the outward-facing conformation. In contrast, distinct "noncatalytic" residues (adjacent to the ion-coordinating residues) control the stability of the outward-facing conformation, but not the catalytic capacity. The helix-breaking signature sequences (GTSLPE) on the α1 and α2 repeats (at the ion-binding core) differ in their folding/unfolding dynamics, while providing asymmetric contributions to transport activities. The present data strongly support the idea that asymmetric preorganization of the ligand-free ion-pocket predefines catalytic reorganization of ion-bound residues, where secondary interactions with adjacent residues couple the alternating access. These findings provide a structure-dynamic basis for ion-coupled alternating access in NCX and similar proteins.
- MeSH
- archeální proteiny chemie genetika metabolismus MeSH
- Escherichia coli genetika metabolismus MeSH
- exprese genu MeSH
- hmotnostní spektrometrie MeSH
- interakční proteinové domény a motivy MeSH
- iontový transport MeSH
- katalytická doména MeSH
- koncentrace vodíkových iontů MeSH
- Methanocaldococcus chemie genetika metabolismus MeSH
- molekulární modely MeSH
- pumpa pro výměnu sodíku a vápníku chemie genetika metabolismus MeSH
- rekombinantní proteiny chemie genetika metabolismus MeSH
- sbalování proteinů MeSH
- sekundární struktura proteinů MeSH
- sekvence aminokyselin MeSH
- sodík chemie metabolismus MeSH
- vápník chemie metabolismus MeSH
- vazba proteinů MeSH
- vodík-deuteriová výměna MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
