氧敏感蛋白的氢氘交换质谱法

Luke  Berry; Angela  Patterson; Natasha  Pence; John W. Peters; Brian  Bothner

doi:10.21769/BioProtoc.2769

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Peer-reviewed

Hydrogen Deuterium Exchange Mass Spectrometry of Oxygen Sensitive Proteins

氧敏感蛋白的氢氘交换质谱法

BB Brian Bothner email

发布: 2018年03月20日第8卷第6期 DOI: 10.21769/BioProtoc.2769 浏览次数: 9042

评审: Vamseedhar RayaproluPaul FinchAnonymous reviewer(s)

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Abstract

The protocol detailed here describes a way to perform hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) on oxygen sensitive proteins. HDX-MS is a powerful tool for studying the protein structure-function relationship. Applying this technique to anaerobic proteins provides insight into the mechanism of proteins that perform oxygen sensitive chemistry. A problem when using HDX-MS to study anaerobic proteins is that there are many parts that require constant movement into and out of an anaerobic chamber. This can affect the seal, increasing the likelihood of oxygen exposure. Exposure to oxygen causes the cofactors bound to these proteins, a common example being FeS clusters, to no longer interact with the amino acid residues responsible for coordinating the FeS clusters, causing loss of the clusters and irreversible inactivation of the protein. To counteract this, a double vial system was developed that allows the preparation of solutions and reaction mixtures anaerobically, but also allows these solutions to be moved to an aerobic environment while shielding the solutions from oxygen. Additionally, movement isn’t limited like it is in an anaerobic chamber, ensuring more consistent data, and fewer errors during the course of the reaction.

Keywords: HDX-MS (HDX-MS)

Mass spectrometry (质谱法)

Anaerobic proteins (厌氧蛋白)

H/D exchange (H/D交换)

Protein dynamics (蛋白质动力学)

Protein-protein interactions (蛋白-蛋白相互作用)

Protein-ligand interactions (蛋白-配体相互作用)

Background

Many oxygen sensitive proteins are required for organisms to thrive in an anoxic environment. Some of these proteins provide an alternative supply of energy to anaerobic microbes through a process known as Flavin-based electron bifurcation (FBEB) (Lubner et al., 2017). FBEB generates reduced ferredoxin, which can be oxidized to produce energy. Proteins that are capable of reducing ferredoxin are of great interest and have been the focus of recent studies using HDX-MS (Demmer et al., 2016; Lubner et al., 2017; Berry et al., 2018). HDX-MS is a powerful technique for investigating protein stability, dynamics, and ligand binding providing information about the relationship between structure and function. HDX-MS uses the intrinsic property of amide hydrogens to exchange with hydrogens in solution to track changes in the structure and dynamics of a protein/protein complex. By preparing buffers with heavy water (D₂O) instead of monoisotopic water (H₂O), amide hydrogens on a protein will exchange with the deuterium in solution. The rate of exchange for a given amino acid is influenced by the stability of hydrogen bonds in the secondary structure, as well as the tertiary and quaternary interactions within a single protein or protein complex. Using mass spectrometry, deuterium incorporation is determined by measuring the shift in isotope distribution between deuterated and non-deuterated samples. HDX-MS has been applied to a large number of proteins and protein complexes across a wide range of conditions. To successfully study these proteins with HDX-MS, it was imperative to establish a means of performing this reaction on the benchtop to avoid heavy traffic into and out of an anaerobic chamber which is time consuming and burdensome. The problem was then how to allow manipulation of the sample while keeping the protein sample anaerobic for an extended period of time in an aerobic environment. To solve this problem, the reaction mixture and protein stock solutions were placed into a double vial system that allowed addition and removal of sample while maintaining strict anaerobic conditions. The logic behind the setup was to create an airlock. Vials are placed under positive pressure with nitrogen gas with a screw cap vial, inside a larger crimp vial that contains reductant. With this double barrier system, small volumes of air can be trapped in the outer vial and do not contact the sample.

Materials and Reagents

Verex^TM vial kit, 9 mm, screw top, polypropylene, 300 μl + PTFE/silicone cap, blue, 1,000/pk (Phenomenex, catalog number: AR0-9991-13 )
Clear glass serum vial with 20 mm crimp top finish, 10 ml, 100/case (DWK Life Sciences, WHEATON, catalog number: 225278 )
Curwood Parafilm M^TM laboratory wrapping film (Bemis, catalog number: PM996 )
Costar Microcentrifuge Tubes 0.65 ml, 500/bag (Corning, catalog number: 3208 )
Onyx Monolithic C18 column, 100 x 2 mm (Phenomenex, catalog number: CH0-8467 )
Alternative column (see Notes for a detailed explanation): Onyx Monolithic C18 column, 100 x 3 mm (Phenomenex, catalog number: CH0-8158 )
Model 1701 and 1702 small RN syringes, 10 μl (26s gauge) and 25 μl (22s gauge), 2” needle point style 2 (Hamilton, catalog numbers: 80030 and 80230 )
Unlined aluminum open-top seals, 20 mm, 1,000/case (DWK Life Sciences, WHEATON, catalog number: 224178-05 )
20 mm stopper, straight plug, ultra-pure (DWK Life Sciences, WHEATON, catalog number: W224100-405 )
200 μl Pipet Tips (VWR, catalog number: 53508-810 )
Liquid nitrogen
Purified ferredoxin (Fd) in 50 mM Ammonium Acetate buffer at pH 6.8 in H₂O (stock concentration 150 μM) from Pf
Note: pH adjusted with 1 N HCl.
Purified NADH-dependent ferredoxin-NADP⁺ oxidoreductase (Nfn) in 20 mM Tris, 150 mM NaCl buffer at pH 8 in H₂O (stock concentration 16.5 mg/ml) from the organism Pyrococcus furiosus (Pf)
Note: pH adjusted with 1 N HCl.
Sodium dithionite (Merck, catalog number: 1065051000 )
Nicotinamide adenine dinucleotide (NAD⁺, Cayman Chemical, catalog number: 16077 , 500 mg)
Nicotinamide adenine dinucleotide (NADH, Cayman Chemical, catalog number: 16078 , 500 mg)
Nicotinamide adenine dinucleotide phosphate (NADP⁺, Cayman Chemical, catalog number: 10004675 , 50 mg)
Nicotinamide adenine dinucleotide phosphate (NADPH, Cayman Chemical, catalog number: 9000743 , 25 mg)
99.5% formic acid (Fisher Scientific, catalog number: A117-50 )
Pepsin from porcine gastric mucosa (Sigma-Aldrich, catalog number: P6887-1G )
Sodium acetate trihydrate (Fisher Scientific, catalog number: S607-500 )
Ammonium acetate, ≥ 99% (Sigma-Aldrich, catalog number: 09689-250G )
37% hydrochloric acid (= 12.1 M) (Merck, catalog number: HX0603-3 )
Sodium hydroxide (Fisher Scientific, catalog number: BP359-500 )
Deuterium oxide (Sigma-Aldrich, catalog number: 151882-100G )
Tris base (Merck, catalog number: 648311-5KG )
Sodium chloride (Fisher Scientific, catalog number: BP358-212 )
HPLC grade water (Fisher Scientific, catalog number: W5-4 )
HPLC grade acetonitrile (Fisher Scientific, catalog number: A998-4 )
Nanopure water (purified in-house using a Millipore Q-Gard 2)
Tris base, NaCl buffer (pH 7.0) in deuterium oxide (see Recipes)
Tris base, NaCl buffer (pH 7.0) in H₂O (see Recipes)
Tris base, NaCl, sodium dithionite buffer (pH 7.0) in H₂O (see Recipes)

Equipment

HPLC stack for separation of peptides generated via pepsin digestion (e.g., 1290 Infinity series HPLC stack manufactured by Agilent Technologies) (Agilent Technologies, model: 1290 Infinity Series )
LC/MS Q-TOF system for sample analysis/data acquisition (e.g., 6538 UHD Accurate-Mass Q-TOF LC/MS manufactured by Agilent Technologies) (Agilent Technologies, model: 6538 UHD Accurate-Mass Q-TOF LC/MS )
Glove box capable of maintaining anaerobic conditions under positive inert gas pressure (e.g., MBraun, model: UNIlab Plus Glove Box Workstation )
Nitrogen tank
20 mm Kebby standard crimper for aluminum seals (Medical Laboratory Supply, catalog number: 2001-00-C01A )
Fisher Scientific isotemp 110 water bath (Fisher Scientific, model: Fisherbrand^TM Isotemp^TM, catalog number: S63077Q )
Note: This product has been discontinued.
Milli-Q purification system (Merck, catalog number: QGARD00D2 )
Pipettes (10 μl and 100 μl) (Eppendorf, catalog numbers: 022478886 and 022478924 )

Software

Microsoft Excel 2016 on Windows 7
UCSF Chimera v. 1.11.2
MassHunter Workstation Software Qualitative Analysis v. B.06.00 (Agilent Technologies)
HDExaminer v. 1.3.0 beta 6 (Sierra Analytics, Inc.)
MassHunter Workstation Software LC/MS Data Acquisition for 6200 series TOF/6500 series Q-TOF v. B.05.01 (Agilent Technologies)
Peptide Analysis Worksheet Freeware Edition (PAWs, ProteoMetrics–freeware edition)
SearchGUI v. 3.2.18 (Compomics)
Peptide Shaker v. 1.16.9 (Compomics)

Procedure

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如何引用

Berry, L., Patterson, A., Pence, N., Peters, J. W. and Bothner, B. (2018). Hydrogen Deuterium Exchange Mass Spectrometry of Oxygen Sensitive Proteins. Bio-protocol 8(6): e2769. DOI: 10.21769/BioProtoc.2769.