Hdx manager

Manufactured by Waters Corporation

Sourced in United Kingdom

HDX Manager is a software tool developed by Waters Corporation to manage and analyze data generated from hydrogen-deuterium exchange (HDX) experiments. It provides a user-friendly interface for processing, visualizing, and interpreting HDX data, which is a widely used technique for studying protein structure and dynamics.

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NanoUPLC HDX Sample Manager (3 citations) Leap HDX Automation Manager (2 citations)

15 protocols using hdx manager

Mapping Protein Deuteration by Mass Spectrometry

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Proteins were diluted to 3.5 μM in phosphate buffered saline, pH 7.4. This stock was used to initiate labelling experiments by diluting 10-fold with deuterated (10 mM sodium phosphate, pD 6.6) aqueous solvent. Initial mapping experiments were performed to assign mass spectra to peptic peptide sequences from IL-33. This was done largely as described39 (link). Briefly, protonated diluted protein was mixed 1:1 with a quench solution (100 mM potassium phosphate, pH 2.55, 0.1 M TCEP, 1 °C), such that the final mixture pH was 2.55. The quenched protein was injected into a Waters HDX Manager with an immobilized pepsin column (2.0 × 30 mm; Poroszyme, Life Technologies), C18 trapping column (VanGuard ACQUITY BEH 2.1 × 5 mm; Waters) and analytical C18 column (1.0 × 100 mm ACQUITY BEH; Waters). Mobile phases were 0.1% formic acid in H₂O (A) and 0.1% formic acid in acetonitrile (B), such that their pH was 2.55. Protein was applied to the pepsin and trapping columns in 100 μl min^–1 buffer A and eluted from the analytical column in a linear gradient of 3–40% B at 40 μL min^–1. Peptide sequences were assigned from MSE fragment data with Protein Lynx Global Server (Waters) 3.0.2 and DynamX 3.0 (Waters). Labeling data was acquired as for sequencing, except the mass spectrometer was set to MS scans only. Peptide-level data were analysed in DynamX and MatLab (Mathworks).

Cohen E.S., Scott I.C., Majithiya J.B., Rapley L., Kemp B.P., England E., Rees D.G., Overed-Sayer C.L., Woods J., Bond N.J., Veyssier C.S., Embrey K.J., Sims D.A., Snaith M.R., Vousden K.A., Strain M.D., Chan D.T., Carmen S., Huntington C.E., Flavell L., Xu J., Popovic B., Brightling C.E., Vaughan T.J., Butler R., Lowe D.C., Higazi D.R., Corkill D.J., May R.D., Sleeman M.A, & Mustelin T. (2015). Oxidation of the alarmin IL-33 regulates ST2-dependent inflammation. Nature Communications, 6, 8327.

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Virus HA Protein Structural Analysis

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Samples were thawed for 5 min on ice and manually injected into a Waters HDX Manager kept at 1°C. Whole-virus HA samples were trapped on a Waters ACQUITY UPLC CSH C18 VanGuard, 130 Å, 1.7 μm, 2.1 mm by 5 mm trap column for 3 min with a flow of solvent A (2% acetonitrile, 0.1% FA, and 0.025% trifluoroacetic acid) at a rate of 150 μl/min. BHA samples were digested online with immobilized pepsin for 5 min and trapped as described previously (51 (link)). Peptides were resolved over a Waters ACQUITY UPLC CSH C18, 130 Å, 1.7 μm, 1 mm by 100 mm column using a 10-min linear gradient of 3 to 50% solvent B (solvent B: 100% acetonitrile and 0.1% FA) and analyzed using a Waters Synapt G2-Si Q-TOF with ion mobility enabled. Source and desolvation temperatures were 70° and 130°C, respectively. The StepWave ion guide settings were tuned to prevent nonuniform gas-phase proton exchange in the source (52 (link)). A series of trap column wash steps were implemented between each injection to minimize carryover (53 (link)).

Benhaim M.A., Mangala Prasad V., Garcia N.K., Guttman M, & Lee K.K. (2020). Structural monitoring of a transient intermediate in the hemagglutinin fusion machinery on influenza virions. Science Advances, 6(18), eaaz8822.

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HDX Mass Spectrometry Analysis of Lysenin

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HDX mass spectrometry was performed using Waters HDX manager composed of an automated sample preparation LEAP robot and a nano-Acquity UPLC coupled to a Synapt G2-Si mass spectrometer. The HDX reaction was initiated by a 13 × dilution into deuterated buffer containing 50 mM Tris, 100 mM NaCl adjusted to pH 9 using NaOD at room temperature for a time course of 20, 100 and 1,000 s for lysenin, whereas for the oligomer the deuteration buffer was supplemented with 0.025% DDM. Samples were sequentially quenched using 45 μl of hydrochloric acid (100 mM) and brought to pH 2.3. The protein was digested in-line using a pepsin immobilized column (Waters) at 20 °C. The generated peptides were trapped on a peptide trap for 3 min for desalting at flow rate of 200 μl min⁻¹ and then separated using a C18 column with a linear gradient 5–80% of acetonitrile and water both supplemented with 0.1% formic acid for 12 min at 0 °C at flow rate 50 μl min⁻¹. Sequence coverage and deuterium uptake were analysed using PLGS and DynamX programmes, respectively.

Podobnik M., Savory P., Rojko N., Kisovec M., Wood N., Hambley R., Pugh J., Wallace E.J., McNeill L., Bruce M., Liko I., Allison T.M., Mehmood S., Yilmaz N., Kobayashi T., Gilbert R.J., Robinson C.V., Jayasinghe L, & Anderluh G. (2016). Crystal structure of an invertebrate cytolysin pore reveals unique properties and mechanism of assembly. Nature Communications, 7, 11598.

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Hydrogen-Deuterium Exchange of Recombinant Coagulation Factors

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rFVIIIFc, rFVIII, and rFc were dialyzed against 10 mM histidine, pH 7.0, 5 mM CaCl₂, 200 mM NaCl and 13.3 g/L sucrose. Deuterium exchange was initiated by diluting each sample ten-fold with deuterated buffer (99.99% D₂O; Cambridge Isotope Laboratories, Andover, MA) to a final volume of 25 μL. After 10 s, 1 min, 10 min, 1 h and 4 h of incubation, the reaction was quenched, and the protein was denatured and reduced by addition of ice-cold quench solution (1:1, v:v) containing 7.5 M guanidinium hydrochloride, 0.2 M Tris (2-carboxyethyl) phosphine (TCEP) and 0.5 M citric acid, resulting in a pH of 2.3. This preparation was digested on an immobilized pepsin column (Life Technologies, Carlsbad, CA) inside a Waters HDX manager with the temperature maintained at 0°C. Eluted peptides were desalted, separated on an HSS T3 C18 HPLC column and introduced into a Synapt G2S mass spectrometer by electrospray ionization. Mass spectra were collected in triplicate for each exchange period. Peptides were identified by ProteinLynx Global Server (Waters, Milford, MA).
HDX data analysis was performed with the DynamX software package (Waters, Milford, MA) and statistical relevance was based on published criteria [25 (link)].

Leksa N.C., Chiu P.L., Bou-Assaf G.M., Quan C., Liu Z., Goodman A.B., Chambers M.G., Tsutakawa S.E., Hammel M., Peters R.T., Walz T, & Kulman J.D. (2017). The structural basis for the functional comparability of Factor VIII and the long-acting variant recombinant Factor VIII Fc fusion protein. Journal of thrombosis and haemostasis : JTH, 15(6), 1167-1179.

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Automated Hydrogen-Deuterium Exchange Mass Spectrometry

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H/DX-MS experiments were performed on a fully automated system equipped with a Leap robot (HTS PAL; Leap Technologies, NC), a Waters ACQUITY M-Class UPLC, a H/DX manager (Waters Corp., Milford, MA) and a Synapt G2-S mass spectrometer (Waters Corp., Milford, MA), as described elsewhere (Zhang et al., 2014 (link)). The protein samples were diluted in a ratio of 1:20 with deuterium oxide containing PBS buffer (pH 7.4) and incubated for 0 s, 10 s, 1 min, 10 min, 30 min or 2 hr. The exchange was stopped by diluting the labeled protein 1:1 in quenching buffer (200 mM Na₂HPO₄ × 2 H₂O, 200 mM NaH₂PO₄ × 2H₂O, 250 mM Tris (2-carboxyethyl)phosphine, 3 M GdmCl, pH 2.2) at 1°C. Digestion was performed on-line using an immobilized Waters Enzymate BEH Pepsin Column (2.1 × 30 mm) at 20°C. Peptides were trapped and separated at 0°C on a Waters AQUITY UPLC BEH C18 column (1.7 µm, 1.0 × 100 mm) by a H₂O to acetonitrile gradient with both eluents containing 0.1% formic acid (v/v). Eluting peptides were subjected to the Synapt TOF mass spectrometer by electrospray ionization. Samples were pipetted by a LEAP autosampler (HTS PAL; Leap Technologies, NC). Data analysis was conducted with the Waters Protein Lynx Global Server PLGs (version 3.0.3) and DynamX (Version 3.0) software package.

Kazman P., Vielberg M.T., Pulido Cendales M.D., Hunziger L., Weber B., Hegenbart U., Zacharias M., Köhler R., Schönland S., Groll M, & Buchner J. (2020). Fatal amyloid formation in a patient’s antibody light chain is caused by a single point mutation. eLife, 9, e52300.

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Time-Resolved Protein Labeling Workflow

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Protein
samples (20 μL) were labeled with the ms2min system, at nine
time points (0.05, 0.15, 0.25, 0.35, 0.5, 1, 5, 30, and 300 s), randomly n = 3. All labeling experiments were performed at 23 °C,
further quenched, and analyzed at 0 °C. A 72 in. long tubing
was used to connect the ms2min system to the digestion/separation
chamber of the Waters HDX Manager. Protein samples were digested online
with a pepsin column (Waters). Buffers used were 40 mM tris hydrochloride,
1 mM TCEP, pH 7.00 in H₂O, 40 mM Tris hydrochloride, and
1 mM TCEP at pD 7.00 in D₂0 and 100 mM potassium phosphate,
pH 2.50 in H₂O, as equilibrium, labeling and quench buffers,
respectively.

Kish M., Subramanian S., Smith V., Lethbridge N., Cole L., Vollmer F., Bond N.J, & Phillips J.J. (2023). Allosteric Regulation of Glycogen Phosphorylase by Order/Disorder Transition of the 250′ and 280s Loops. Biochemistry, 62(8), 1360-1368.

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Hydrogen-Deuterium Exchange Mass Spectrometry Protocol

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To determine HDX-MS, a HDX-MS system from Waters, consisting of a Synapt G2-Si MS, ACQUITY M-Class UPLC, HDX Manager, and a LEAP HDX-2 automation platform, was used as described previously (54 (link), 88 (link)). Measurements were performed with an initial protein concentration of 30 μM which was diluted in a 1:20 ratio into PBS with D₂O. Samples were incubated for 10, 60, 600, 1800, and 7200 s. As 0 s, the experiment was performed without Deuterium. HD exchange was stopped with a quenching solution (200 mM Na₂HPO₄, 200 mM KH₂PO₄, 4 M GdmCl, pH 2.3) in a 1:1 ratio at 1 °C. The sample was directly transferred on a digestive Pepsin Column (Waters Enzymate BEH Pepsin Column 2.1 mm × 30 mm). The pepsin digestion took place at 0 °C. Peptides separated by chromatography using an ACQUITY UPLC BEH C18 1.7 μM VanGuard Pre-Column 2. 1 × 5 mm, followed by an analytical column (ACQUITY UPLC BEH C18 1.7 μM 1.0 × 100 mm) before peptides were measured by MS. Analysis of the HDX data was done with DynamX (Version 3.0).

Strauch A., Rossa B., Köhler F., Haeussler S., Mühlhofer M., Rührnößl F., Körösy C., Bushman Y., Conradt B., Haslbeck M., Weinkauf S, & Buchner J. (2022). The permanently chaperone-active small heat shock protein Hsp17 from Caenorhabditis elegans exhibits topological separation of its N-terminal regions. The Journal of Biological Chemistry, 299(1), 102753.

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HDX-MS Analysis of Chl1 Protein

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HDX-MS was performed using a Waters HDX manager. Samples were prepared by 10-fold dilutions from 5 μM Chl1 protein in deuterated or nondeuterated buffer containing 150 mM NaCl, 50 mM HEPES pH 7.5, 0.5 mM TCEP. The pH of the sample was reduced to 2.3 and an in-line pepsin-immobilized column at 20°C was used for protein digestion. For labelling experiments, apo protein was incubated for 10 s, 100 s, and 1000 s at room temperature. All HDX-MS experiments were performed in triplicate. Sequence coverage and deuterium uptake were analysed by using ProteinLynx Global Server (Waters) and DynamX (Waters) programs, respectively. For mass correction, Leucine enkephalin at a continuous flow rate of 5 μl min⁻¹ was sprayed. All sample preparation and sample loading performed manually.

Hodáková Z., Nans A., Kunzelmann S., Mehmood S., Taylor I., Uhlmann F., Cherepanov P, & Singleton M.R. (2021). Structural characterisation of the Chaetomium thermophilum Chl1 helicase. PLoS ONE, 16(5), e0251261.

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Online Protein Digestion and Peptide Analysis

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A Waters M-Class coupled with HDX Manager (equipped with a 50 μL loop) was used to perform protein digestion followed by peptide trapping and elution. Buffers A and B consisted of 0.1% (v/v) formic acid and 0.1% (v/v) formic acid in 100% acetonitrile, respectively. An Enzymate Pepsin Column (Waters, 300 Å, 5 μm, 2.1 × 30 mm) was used for online digestion at 15 °C. Digestion and subsequent trapping were performed for 3 minutes at a flow of 100 μL/min. All peptides were desalted via reverse-phase trap (Waters Protein BEH C4 VanGuard Pre-column, 300 Å, 1.7 μm, 2.1 × 5 mm) and separated on a C18 column (Waters BEH C18 Column, 130 Å, 1.7 μm, 1 × 100 mm) at 1 °C using an isocratic gradient from 3–40% buffer B at 40 μL/min for 7 minutes. Following peptide digestion, a pepsin wash solution consisting of 2 M GuHCl, 4% ACN, 0.8% (v/v) formic acid, was injected to minimize carryover. Blank injections were performed after each sample injection to ensure that low carryover was maintained.

Blazeck J., Karamitros C.S., Ford K., Somody C., Qerqez A., Murray K., Burkholder N.T., Marshall N., Sivakumar A., Lu W.C., Tan B., Lamb C., Tanno Y., Siddiqui M.Y., Ashoura N., Coma S., Zhang X.M., McGovern K., Kumada Y., Zhang Y.J., Manfredi M., Johnson K.A., D’Arcy S., Stone E, & Georgiou G. (2022). Bypassing evolutionary dead ends and switching the rate-limiting step of a human immunotherapeutic enzyme. Nature catalysis, 5(10), 952-967.

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Detailed HDX-MS Proteomics Protocol

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Data were collected using the HDX Manager (Waters, U.K.) set at 0.1°C in-line with a SYNAPT G2-Si HDMS (Waters, U.K.) fitted with an ETD ion source block. In-line peptic digestion was conducted using an Enzymate BEH Pepsin 2.1 × 30 mm Column (Waters, U.K.) at 15°C, where protein was digested for 2 min. Peptides were eluted onto an Acquity 1.7 µm particle, 100 mm × 1 mm C18 UPLC column (Waters, U.K.) in buffer A (0.1% formic acid) with a 12 min 5–36% gradient of buffer B (100% acetonitrile and 0.1% formic acid). Data were acquired over a mass range of 300–2000 m/z with a spray voltage of 2.6 kV.

Masson G.R., Maslen S.L, & Williams R.L. (2017). Analysis of phosphoinositide 3-kinase inhibitors by bottom-up electron-transfer dissociation hydrogen/deuterium exchange mass spectrometry. Biochemical Journal, 474(11), 1867-1877.

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