High-throughput hydrogen deuterium exchange mass spectrometry (HDX-MS) coupled with subzero-temperature ultrahigh pressure liquid chromatography (UPLC) separation for complex sample analysis

Fang, Mulin; Wang, Zhe; Cupp‐Sutton, Kellye A.; Welborn, Thomas; Smith, Kenneth; Wu, Si

doi:10.1016/j.aca.2020.11.022

Cited by 22 publications

(48 citation statements)

References 28 publications

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“…Sample isolation should be conducted under quench conditions (pH 2.5, 0°C) to reduce intrinsic exchange rates. Specialized liquid chromatography (LC) operating at sub‐zero temperatures may also be employed to improve separation 31,32 . Ideally, in vivo HDX‐MS should be performed without overexpression to preserve the relative protein concentrations, which may be possible with future optimization of the back exchange levels.…”

Section: Discussionmentioning

confidence: 99%

“…Specialized liquid chromatography (LC) operating at sub-zero temperatures may also be employed to improve separation. 31,32 Ideally, in vivo HDX-MS should be performed without overexpression to preserve the relative protein concentrations, which may be possible with future optimization of the back exchange levels. To facilitate rapid isolation under quench conditions, we are in the process of developing a purification tag that functions at low pH.…”

Section: Hdx Protocol Considerationsmentioning

confidence: 99%

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Development of in vivo HDX‐MS with applications to a TonB‐dependent transporter and other proteins

et al. 2022

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Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful tool that monitors protein dynamics in solution. However, the reversible nature of HDX labels has largely limited the application to in vitro systems. Here, we describe a protocol for measuring HDX-MS in living Escherichia coli cells applied to BtuB, a TonB-dependent transporter found in outer membranes (OMs). BtuB is a convenient and biologically interesting system for testing in vivo HDX-MS due to its controllable HDX behavior and large structural rearrangements that occur during the B12 transport cycle. Our previous HDX-MS study in native OMs provided evidence for B12 binding and breaking of a salt bridge termed the Ionic Lock, an event that leads to the unfolding of the amino terminus. Although purified OMs provide a more native-like environment than reconstituted systems, disruption of the cell envelope during lysis perturbs the linkage between BtuB and the TonB complex that drives B12 transport. The in vivo HDX response of BtuB's plug domain (BtuBp) to B12 binding corroborates our previous in vitro findings that B12 alone is sufficient to break the Ionic Lock. In addition, we still find no evidence of B12 bindinginduced unfolding in other regions of BtuBp that could enable B12 passage. Our protocol was successful in reporting on the HDX of several endogenous E. coli proteins measured in the same measurement. Our success in performing HDX in live cells opens the possibility for future HDX-MS studies in a native cellular environment. IMPORTANCEWe present a protocol for performing in vivo HDX-MS, focusing on BtuB, a protein whose native membrane environment is believed to be mechanistically

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Section: Discussionmentioning

confidence: 99%

Section: Hdx Protocol Considerationsmentioning

confidence: 99%

Development of in vivo HDX‐MS with applications to a TonB‐dependent transporter and other proteins

et al. 2022

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“…More recent studies have demonstrated effective LC separation of an entire deuterated Echerichia coli lysate digest separated at −10 °C using a 90 min gradient with only moderate back exchange. 191 …”

Section: Measuring Hydrogen Exchangementioning

confidence: 99%

Advances in Hydrogen/Deuterium Exchange Mass Spectrometry and the Pursuit of Challenging Biological Systems

et al. 2021

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Solution-phase hydrogen/deuterium exchange (HDX) coupled to mass spectrometry (MS) is a widespread tool for structural analysis across academia and the biopharmaceutical industry. By monitoring the exchangeability of backbone amide protons, HDX-MS can reveal information about higher-order structure and dynamics throughout a protein, can track protein folding pathways, map interaction sites, and assess conformational states of protein samples. The combination of the versatility of the hydrogen/deuterium exchange reaction with the sensitivity of mass spectrometry has enabled the study of extremely challenging protein systems, some of which cannot be suitably studied using other techniques. Improvements over the past three decades have continually increased throughput, robustness, and expanded the limits of what is feasible for HDX-MS investigations. To provide an overview for researchers seeking to utilize and derive the most from HDX-MS for protein structural analysis, we summarize the fundamental principles, basic methodology, strengths and weaknesses, and the established applications of HDX-MS while highlighting new developments and applications.

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“…Unlike other peptide-based methods, in which trypsin is the protease of choice, many HDX-MS studies use pepsin for digestion due to its higher efficiency in acidic conditions, which are necessary to minimise back-exchange. [76][77][78] HDX-MS provides useful information for integrative structural biology applications because the rate of amide hydrogen exchange within any given residue of the protein backbone is dependent upon four key factors. These factors are pH, temperature, solvent accessibility and hydrogen bonding.…”

Section: Hydrogen Deuterium Exchange-mass Spectrometry (Hdx-ms)mentioning

confidence: 99%

Integration of Mass Spectrometry Data for Structural Biology

2021

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Mass spectrometry (MS) is increasingly being used to probe the structure and dynamics of proteins and the complexes they form with other macromolecules. There are now several specialised MS methods each with unique sample preparation, data acquisition and data processing protocols. Collectively these methods are referred to as structural MS and include, crosslinking-, hydrogen deuterium exchange-, hydroxyl radical footprinting-native, ion mobility-and top-down MS. Each of these provides a unique type of structural information, ranging from composition and stoichiometry through to residue level proximity and solvent accessibility. Structural MS has proved particularly beneficial in studying protein classes for which analysis by classic structural biology techniques proves challenging, such as glycosylated or intrinsically disordered proteins. To capture the structural details for a particular system, especially larger multiprotein complexes, more than one structural MS method with other structural and biophysical techniques is often required. Key to integrating these diverse data are computational strategies and software solutions to facilitate this process.We provide a background to the structural MS methods and briefly summarise other structural methods and how these are combined with MS. We then describe current state of the art approaches for the integration of structural MS data for structural biology. We quantify how often these methods are used together and provide examples where such combinations have been fruitful. To illustrate the power of integrative approaches, we discuss progress in solving the structures of the proteasome and the nuclear pore complex. We also discuss how information from structural MS, particularly pertaining to protein dynamics is not currently utilised in integrative workflows and how such information can provide a more accurate picture of the systems studied. We conclude by discussing new developments in the MS and computational fields that will further enable in-cell structural studies.

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High-throughput hydrogen deuterium exchange mass spectrometry (HDX-MS) coupled with subzero-temperature ultrahigh pressure liquid chromatography (UPLC) separation for complex sample analysis

Cited by 22 publications

References 28 publications

Development of in vivo HDX‐MS with applications to a TonB‐dependent transporter and other proteins

Development of in vivo HDX‐MS with applications to a TonB‐dependent transporter and other proteins

Advances in Hydrogen/Deuterium Exchange Mass Spectrometry and the Pursuit of Challenging Biological Systems

Integration of Mass Spectrometry Data for Structural Biology

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