Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments

Masson, Glenn R.; Burke, John E.; Ahn, Natalie G.; Anand, Ganesh S.; Borchers, Christoph H.; Brier, Sébastien; Bou-Assaf, George M.; Engen, John R.; Englander, S. Walter; Faber, Johan H.; Garlish, Rachel A.; Griffin, Patrick R.; Gross, Michael L.; Guttman, Miklós; Hamuro, Yoshitomo; Heck, Albert J. R.; Houde, Damian J.; Iacob, Roxana E.; Jørgensen, Thomas J. D.; Kaltashov, Igor A.; Klinman, Judith P.; Konermann, Lars; Man, Petr; Mayne, Leland; Pascal, Bruce D.; Reichmann, Dana; Skehel, Mark; Snijder, Joost; Strutzenberg, Timothy S.; Underbakke, Eric S.; Wagner, Cornelia; Wales, Thomas E.; Walters, Benjamin T.; Weis, David D.; Wilson, Derek J.; Wintrode, Patrick L.; Zhang, Zhongqi; Zheng, Jie; Schriemer, David C.; Rand, Kasper D.

doi:10.1038/s41592-019-0459-y

Cited by 499 publications

(510 citation statements)

References 58 publications

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“…The full details of H/D exchange for all peptides are shown in Source data, with statistics described in Table EV1. All HDX-MS data was analyzed according to the guidelines set out by the IC-HDX-MS community [60].…”

Section: Hdx-ms Data Analysismentioning

confidence: 99%

Characterization of the c10orf76‐PI4KB complex and its necessity for Golgi PI4P levels and enterovirus replication

et al. 2019

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The lipid kinase PI4KB, which generates phosphatidylinositol 4phosphate (PI4P), is a key enzyme in regulating membrane transport and is also hijacked by multiple picornaviruses to mediate viral replication. PI4KB can interact with multiple protein binding partners, which are differentially manipulated by picornaviruses to facilitate replication. The protein c10orf76 is a PI4KB-associated protein that increases PI4P levels at the Golgi and is essential for the viral replication of specific enteroviruses. We used hydrogendeuterium exchange mass spectrometry to characterize the c10orf76-PI4KB complex and reveal that binding is mediated by the kinase linker of PI4KB, with formation of the heterodimeric complex modulated by PKA-dependent phosphorylation. Complexdisrupting mutations demonstrate that PI4KB is required for membrane recruitment of c10orf76 to the Golgi, and that an intact c10orf76-PI4KB complex is required for the replication of c10orf76dependent enteroviruses. Intriguingly, c10orf76 also contributed to proper Arf1 activation at the Golgi, providing a putative mechanism for the c10orf76-dependent increase in PI4P levels at the Golgi.

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Section: Hdx-ms Data Analysismentioning

confidence: 99%

Characterization of the c10orf76‐PI4KB complex and its necessity for Golgi PI4P levels and enterovirus replication

et al. 2019

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“…Changes in any peptide at any time point greater than specified cut-offs (both 7% and 0.4 Da between conditions for the Q572* mutant versus wild-type and both 7% and 0.4 Da between conditions for the PDGFR pY dose experiment) and with an unpaired t-test value of p<0.05 was considered significant. The full set of HDX-MS data is presented in Source Data 1, with all HDX-MS analysis statistics shown in Table S1 as suggested by the guidelines set out in (Masson et al, 2019).…”

Section: Mass Analysis Of Peptide Centroids and Measurement Of Deutermentioning

confidence: 99%

Defining how oncogenic and developmental mutations ofPIK3R1alter the regulation of class IA phosphoinositide 3-kinases

Dornan

Stariha

Rathinaswamy

et al. 2019

Preprint

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The class IA PI3Ks are key signalling enzymes composed of a heterodimer of a p110 catalytic subunit and a p85 regulatory subunit, with PI3K mutations being causative of multiple human diseases including cancer, primary immunodeficiencies, and developmental disorders.Mutations in the p85α regulatory subunit encoded by PIK3R1 can both activate PI3K through oncogenic truncations in the iSH2 domain, or inhibit PI3K through developmental disorder mutations in the cSH2 domain. Using a combined biochemical and hydrogen deuterium exchange mass spectrometry approach we have defined the molecular basis for how these mutations activate both the p110α/p110δ catalytic subunits. We find that the oncogenic Q572* truncation of PIK3R1 disrupts all inhibitory inputs, with p110α being hyper-activated compared to p110δ. In addition, we find that the R649W mutation in the cSH2 of PIK3R1 decreases sensitivity to activation by receptor tyrosine kinases. This work reveals novel insight into isoform specific regulation of p110s by p85α.Keywords: PI3K, p110, p85, phosphoinositide 3-kinase, PI3K-Akt, HDX-MS, hydrogen exchange, phosphoinositides, PIK3CA, PIK3R1Defining the p85 mechanisms that mediate the inhibition and activation of PI3K Highlights-An oncogenic variant of p85α, Q572*, leads to hyper-activation of p110α compared to p110δ -HDX-MS revealed that Q572* leads to disruption of all inhibitory interfaces in p110α -A SHORT syndrome mutation in p85α leads to decreased sensitivity to RTKs for p110α/δ Defining the p85 mechanisms that mediate the inhibition and activation of PI3K

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“…What were once considered pipedreams of structural biology are now within reach. Furthermore, as HDX‐MS becomes used by a larger population of researchers, significant efforts have been made across the field to standardize experimental procedures and data processing and reporting practices …”

Section: Resultsmentioning

confidence: 99%

“…Compared to other high-resolution structural techniques, information about protein conformation can be obtained using HDX-MS for samples spanning a range of sizes and complexities, provided one's analyte of interest is homogeneous in composition and well-behaved in solution. 3 While HDX can also be probed by NMR with residue level resolution, this approach is generally restricted to small soluble proteins that are amenable to solution state NMR analysis. 2,4 By contrast, mass spectrometers have no inherent size limit and have been used to study systems as large and complex as intact ribosomal complexes.…”

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confidence: 99%

Bridging protein structure, dynamics, and function using hydrogen/deuterium‐exchange mass spectrometry

2019

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Much of our understanding of protein structure and mechanistic function has been derived from static high‐resolution structures. As structural biology has continued to evolve it has become clear that high‐resolution structures alone are unable to fully capture the mechanistic basis for protein structure and function in solution. Recently Hydrogen/Deuterium‐exchange Mass Spectrometry (HDX‐MS) has developed into a powerful and versatile tool for structural biologists that provides novel insights into protein structure and function. HDX‐MS enables direct monitoring of a protein's structural fluctuations and conformational changes under native conditions in solution even as it is carrying out its functions. In this review, we focus on the use of HDX‐MS to monitor these dynamic changes in proteins. We examine how HDX‐MS has been applied to study protein structure and function in systems ranging from large, complex assemblies to intrinsically disordered proteins, and we discuss its use in probing conformational changes during protein folding and catalytic function. Statement for a Broad Audience The biophysical and structural characterization of proteins provides novel insight into their functionalities. Protein motions, ranging from small scale local fluctuations to larger concerted structural rearrangements, often determine protein function. Hydrogen/Deuterium‐exchange Mass Spectrometry (HDX‐MS) has proven a powerful biophysical tool capable of probing changes in protein structure and dynamic protein motions that are often invisible to most other techniques.

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Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments

Cited by 499 publications

References 58 publications

Characterization of the c10orf76‐PI4KB complex and its necessity for Golgi PI4P levels and enterovirus replication

Characterization of the c10orf76‐PI4KB complex and its necessity for Golgi PI4P levels and enterovirus replication

Defining how oncogenic and developmental mutations ofPIK3R1alter the regulation of class IA phosphoinositide 3-kinases

Bridging protein structure, dynamics, and function using hydrogen/deuterium‐exchange mass spectrometry

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