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Improving spectral validation rates in hydrogen-deuterium exchange data analysis.

ABSTRACT: HX-MS data generated to support the testing and development of the HX data analysis applications in Mass Spec Studio.

INSTRUMENT(S): Orbitrap Fusion Lumos, TripleTOF 5600

ORGANISM(S): Homo Sapiens (human) Oryctolagus Cuniculus (rabbit)

SUBMITTER: Shaunak Raval

LAB HEAD: David Schriemer

PROVIDER: PXD022831 | Pride | 2021-02-22

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	0.5ug_Eg5_0.5ug_Nepii_20_D2O_10min_rep1.wiff	Wiff
	0.5ug_Eg5_0.5ug_Nepii_20_D2O_10min_rep1.wiff.scan	Wiff
	0.5ug_Eg5_0.5ug_Nepii_20_D2O_10min_rep2.wiff	Wiff
	0.5ug_Eg5_0.5ug_Nepii_20_D2O_10min_rep2.wiff.scan	Wiff
	0.5ug_Eg5_0.5ug_Nepii_20_D2O_10min_rep3.wiff	Wiff

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Publications

Improving Spectral Validation Rates in Hydrogen-Deuterium Exchange Data Analysis.

Raval Shaunak S Sarpe Vladimir V Hepburn Morgan M Crowder D Alex DA Zhang Terry T Viner Rosa R Schriemer David C DC

Analytical chemistry 20210216 9

The data analysis practices associated with hydrogen-deuterium exchange mass spectrometry (HX-MS) lag far behind that of most other MS-based protein analysis tools. A reliance on external tools from other fields and a persistent need for manual data validation restrict this powerful technology to the expert user. Here, we provide an extensive upgrade to the HX data analysis suite available in the Mass Spec Studio in the form of two new apps (HX-PIPE and HX-DEAL), completing a workflow that provi ...[more]

PMID: 33592142

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Epitope Mapping of Human Polyclonal Antibodies to the fHbp Antigen of a Neisseria Meningitidis Vaccine by Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS)

Project description:Antigen-antibody interactions play a key role in the immune response post vaccination and the mechanism of action of antibody-based biopharmaceuticals. 4CMenB is a multicomponent vaccine against Neisseria meningitidis serogroup B in which factor H binding protein (fHbp) is one of the key antigens. In this study, we use hydrogen/deuterium exchange mass spectrometry (HDX-MS) to identify epitopes in fHbp recognized by polyclonal antibodies (pAb) from two human donors (HDs) vaccinated with 4CMenB. Our HDX-MS data reveal several epitopes recognized by the complex mixture of human pAb. Furthermore, we show that the pAb from the two HDs recognize the same epitope regions. Epitope mapping of total pAb and purified fHbp-specific pAb from the same HD reveals that the two antibody samples recognize the same main epitopes, showing that HDX-MS based epitope mapping can, in this case at least, be performed directly using total IgG pAb samples that have not undergone Ab-selective purification. Two monoclonal antibodies (mAb) were previously produced from B-cell repertoire sequences from one of the HDs and used for epitope mapping of fHbp with HDX-MS. The epitopes identified for the pAb from the same HD in this study, overlap with the epitopes recognized by the two individual mAbs. Overall, HDX-MS epitope mapping appears highly suitable for simultaneous identification of epitopes recognized by pAb from human donors and to thus both guide vaccine development and study basic human immunity to pathogens, including viruses.

2024-05-24 | PXD046572 | Pride

Epitope Mapping of Human Polyclonal Antibodies to the fHbp Antigen of a Neisseria Meningitidis Vaccine by Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) - part 2

2024-05-22 | PXD047712 | Pride

Automating data analysis for hydrogen/deuterium exchange mass spectrometry using data-independent acquisition methodology

Project description:We developed an HX-MS2 strategy that supports the acquisition of CID-generated fragment ions alongside their peptide precursors. The approach enables true auto-validation of HX data by mining the rich set of deuterated fragments, using the extra dimension of data to simultaneously confirm the peptide ID and authenticate (even correct) MS1-based deuteration calculations. The high redundancy provided by the fragments generates an opportunity to determine the confidence of deuterium calculations using a combinatorial strategy. The approach requires DIA-based acquisition methods that are available on most MS platforms, making the switch to HX-MS2 straightforward. Considerable time is saved through auto-validation and more importantly, more complex samples can now be characterized and at higher throughput, as illustrated in a drug binding analysis of the ultralarge kinase DNA-PKcs, isolated directly from mammalian cells.

2023-09-08 | PXD045012 | Pride

Interdomain-linker control conformational transitions in the SLC23 elevator transporter UraA

Project description:Uptake of nucleobases and ascorbate is an essential process in all living organisms mediated by SLC23 transport proteins. These transmembrane carriers operate via the elevator alternating access mechanism and are composed of two rigid domains whose relative motion drives transport. The lack of large conformational changes within these domains suggests that the interdomain-linkers act as flexible tethers. Here, we show that interdomain-linkers are not mere tethers but have a key regulatory role in dictating the conformational space of the transporter and defining the rotation axis of the mobile transport domain. By resolving a wide inward-open conformation of the SLC23 elevator transporter UraA and combining biochemical studies using a synthetic nanobody as conformational probe with hydrogen-deuterium exchange mass spectrometry, we demonstrate that interdomain-linkers control the function of transport proteins by influencing substrate affinity and transport rate. These findings open the possibility to allosterically modulate the activity of elevator proteins by targeting their linkers.

2024-10-17 | PXD054394 | Pride

The HisRS-like domain of GCN2 is a pseudoenzyme that can bind uncharged tRNA - HX-MS Data

Project description:GCN2 is a stress response kinase that phosphorylates the translation initiation factor eIF2to inhibit general protein synthesis when activated by uncharged tRNA and stalled ribosomes. The presence of a HisRS-like domain in GCN2, normally associated with the ability to bind and aminoacylate tRNAs, led to the hypothesis that eIF2 kinase activity is regulated by the direct binding of this domain to uncharged tRNA. Here we solved the structure of the HisRS-like domain in the context of full-length GCN2 by cryoEM. Structure and function analysis shows the HisRS-like domain of GCN2 has lost tRNA charging, ATP binding, and histidine binding activity but retains the ability to bind tRNA. Hydrogen deuterium exchange mass spectrometry (HX-MS), site-directed mutagenesis and computational docking experiments support a tRNA binding model that overlaps with but is partially shifted from that employed by bona fide HisRS enzymes. These results demonstrate that the HisRS-like domain of GCN2 is a pseudoenzyme and advance our understanding of GCN2 regulation and function.

2024-02-01 | PXD049090 | Pride

Timeline of changes in spike conformational dynamics in emergent SARS-CoV-2 variants reveal progressive stabilization of trimer stalk with altered NTD dynamics

Project description:SARS-CoV-2 emergent variants are characterized by increased viral fitness and each shows multiple mutations predominantly localized to the spike (S) protein. Here, amide hydrogen/deuterium exchange mass spectrometry has been applied to track changes in S dynamics from multiple SARS-CoV-2 variants. Our results highlight large differences across variants at two loci with impacts on S dynamics and stability. A significant enhancement in stabilization first occurred with the emergence of D614G S followed by smaller, progressive stabilization in subsequent variants. Stabilization preceded altered dynamics in the N-terminal domain, wherein Omicron BA.1 S showed the largest magnitude increases relative to other preceding variants. Changes in stabilization and dynamics resulting from S mutations detail the evolutionary trajectory of S in emerging variants. These carry major implications for SARS-CoV-2 viral fitness and offer new insights into variant-specific therapeutic development.

2023-03-14 | PXD040717 | Pride

Kinetic Analysis of DNA Dependent Protein Kinase Catalytic Subunit

Project description:We investigated the dynamics of protein kinase DNA dependent protein kinase catalytic subunit (DNA-PKcs) through hydrogen deuterium exchange mass spectrometry. Over 1100 peptides could be monitored throughout the kinetics experiment, showing the utility of our nanospray HX-MS configuration for ultra-large proteins such as DNA-PKcs. To match the structurally unassigned helices to sequence from the large disordered regions of DNA-PKcs (a.a 2577-2773), we used integrative modelling. Two sequence assignments were possible. The cluster of models with the helix assignment towards the N-terminal side was favored by reduced kinetics of exchange as measured by HX-MS.

2019-12-10 | PXD016595 | Pride

Structural Premise of Selective Deubiquitinase USP30 Inhibition by Small-Molecule Benzosulfonamides

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2023-10-24 | PXD041582 | Pride

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