Mobility Capillary Electrophoresis Restrained Modelling Method for

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

Mobility Capillary Electrophoresis Restrained Modelling Method for Protein Structure Analysis in Mixtures Rongkai Zhang, Haimei Wu, Muyi He, Wenjing Zhang, and Wei Xu J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.9b01148 • Publication Date (Web): 26 Feb 2019 Downloaded from http://pubs.acs.org on February 27, 2019

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The Journal of Physical Chemistry

Mobility Capillary Electrophoresis Restrained Modelling Method for Protein Structure Analysis in Mixtures Rongkai Zhang,1# Haimei Wu,1# Muyi He,1 Wenjing Zhang1 and Wei Xu1*

School of Life Science, Beijing Institute of Technology, Beijing 100081, China

1

*Corresponding Author: Wei Xu School of Life Science Beijing Institute of Technology Haidian, Beijing, 100081, China Email: [email protected] Web: http://www.escience.cn/people/weixu #Equal

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Abstract: Protein stereo structure analysis in mixtures still remains challenging, especially large-scale analysis such as in proteomics. With the capability of measuring the hydrodynamic radius of ions in liquid phase, mobility capillary electrophoresis (MCE) has been applied to study the structure of peptides. In this study, MCE was extended for protein mixture separation and their corresponding hydrodynamic radius analyses. After ellipsoid approximation, results obtained by MCE experiments were then used as a restrain in molecular dynamics (MD) simulations to predict the most probable structure of each protein. Besides a three-protein mixture, mixture of disulfide bond reduced insulin was also studied by this MCE restrained modelling method. Results obtained by this method agree with literatures, and mass spectrometry experiments were also carried out to confirm our findings.


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1. Introduction The biological function of a protein depends on its structure. Therefore, great efforts have been made to resolve the structure of proteins. For example, mass spectrometry (MS) and tandem MS based proteomics methods have been developed to analyze proteins in large scale, and mainly the primary sequence and post-translational modifications (PTM) could be obtained.1-3 On the other hand, high resolution protein stereo structure could be resolved using techniques, including nuclear magnetic resonance (NMR), X-ray crystallography and cryo-electron microscopy (cryo-EM).4-8 These high resolution techniques typically require high protein concentration and complicated sample preparation processes, such as labeling and crystallization.9-11 Although MS based methods could analyze trace amount of proteins in mixtures, very limited protein stereo structure information could be obtained. Taylor dispersion analysis (TDA) is a simple method to determine the diffusion coefficients (D) of a molecule under the Laminar flow in a thin glass tube developed by Taylor and Aris in the 1950s,12-14 and then used in conjunction with the Stokes equation to solve the hydrodynamic radius (Rh) of a molecule.15 By applying a constant pressure, analytes (solute) diffuses in the solvent. Analyte concentration depends on the tube diameter, tube length and time. Analyte diffusion in the axial direction is considered, and the hydrodynamic radius (Rh) of a molecule is a function of its diffusion time t and variance σ, which associated with the peak width. At present, TDA is generally conducted on capillary electrophoresis instruments, and has become one of the commonly used methods in the size analyses of proteins 16, 17 and nanoparticles.18, 19 Capillary electrophoresis (CE) refers to a type of separation technology in liquid phase through a fused silica capillary, which has very good performances in the separation of mixtures.20-22 Analytes could be rapidly separated according to their different mobilities in a combination of electroosmotic flow (EOF) and a high-voltage DC electric field. In addition to mixture separations, CE could also be used for studying protein-protein interactions23, 24 and monitoring protein conformational dynamics in solution. 25, 26 Previously, we have developed a method named as mobility capillary electrophoresis (MCE), 27 which combines the concepts of capillary electrophoresis and ion mobility spectroscopy.28-32 The use of MCE in combination with molecular dynamics (MD) simulations had been successfully used in peptide conformation predictions.27 In this work, we have extended the application of this method in separation and structure analyses for protein mixture. As a variance of CE, it is possible in the future to combine MCE with MS, and applied in proteomics for large scale protein sequence, PTM and stereo structure analyses.



2. Materials and methods 2.1 Chemicals and Reagents Cytochrome c, lysozyme, ribonuclease A (RNaseA), insulin, Tris (2-carboxyethyl) phosphine (TCEP) were purchased from Sigma-Aldrich (St. Louis, MO). Dimethyl sulfoxide (DMSO), phenol, sodium chloride (NaCl), ammonium acetate, hydrochloric acid (HCl) and sodium hydroxide (NaOH) were obtained from Beijing Chemical Works (Beijing, China). Formic acid was purchased from Fisher Scientific (Fair Lawn, NJ, USA). Deionized water was purchased from Wahaha Co. (Hangzhou, China). Protein sample solutions were prepared in water as stock solutions at the concentration of 10 mg/mL and diluted to final concentrations in the corresponding running buffer in each experiment. 2.2 Capillary electrophoresis All CE experiments were performed on a Lumex CE system (model Capel 105 M, St. Petersburg, Russia) equipped with a UV detector. The fused silica capillary with a dimension of 75 μm i.d. (360 μm o.d.) × 50 cm (40 cm to the detection window) were purchased from Sino Sumtech (Hebei, China). A new coated (coated with poly N-isopropyl-acrylamide, PAM) capillary was cleaned by rinsing it with water for 30 min before use. Cytochrome, lysozyme, ribonuclease A were analyzed in a condition that a pH neutral solution containing 2 mM NaCl in water (pH 7.0, η = 0.89 mPa·S). Protein mixture samples were diluted to a final concentration of 2 mg/mL, and 0.5% DMSO was added as the neutral marker. Separations were performed by applying a 50 mbar driving pressure and a -15 kV DC voltage. When analyzing insulin, water (with 0.1% FA, pH 3.0, η = 0.89 mPa·S) was used as the running buffer, and uncoated capillaries were used. In reduction of disulfide bond in insulin, Tris(2-carboxyethyl) phosphine (TCEP) solution(100mmol/L) was added in insulin sample as the reducing agent, making the final insulin concentration of 2 mg/ml (~0.35 mmol/L) and TCEP molar concentration 10 times that of insulin, 0.5% phenol (1 mol/L) was also added in the sample as a neutral marker in acidic conditions. A 50 mbar pressure was also applied while the separation voltage was tuned to -20 kV. The working temperature was set at 25 °C, and detection wavelength of the UV detector was 214 nm. Between runs, the capillary was rinsed sequentially with deionized water and running buffer for 2 mins. Samples were injected by applying a 50 mbar pressure for 5 s. 2.3 Mass spectrometry detection A Bruker HCT ion trap mass spectrometer (Bruker Daltonics Inc., MA, Germany) was used for the analyses of reduced insulin samples. Solutions containing 0.05 mg/ml insulin were prepared with water (0.1% FA, pH 3.0), 100 mmol/L Tris(2-carboxyethyl)


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phosphine (TCEP) was also added, making the final molar concentration 10 times to insulin. The samples were electrosprayed via nanoESI glass capillaries (i.d. = 0.84 mm, o.d. = 1.5 mm, length = 80 mm). MS experiments settings were as follows: capillary voltage 1000 V; maximum accumulation time 50 ms; flow rate of nitrogen drying gas 10 L/min and the temperature of the capillary 150 °C. Data was collected in positive ion mode with a mass range from 300 to 2000. 2.4 Molecular dynamics simulation MD simulations were conducted with the GROMOS 54A7 force field 33 in GROMACS 2016.1. 34 The initial structure of a protein was obtained from the RCSB Protein Data Bank (PDB). Protein and their PDB ID are as follows: cytochrome c (1AKK); lysozyme (1GXV); ribonuclease A (1AQP); insulin (1GUJ). The cysteines on the native insulin A chain and B chain were linked by a disulfide bond; while a hydrogen atom was introduced on the cysteine to form a free sulfhydryl group after the reduced reaction. pKa calculations were performed with PROPKA35, 36 to evaluate protonation states of each titratable residue, and then the charge states of proteins were obtained at different pHs (7+ for cytochrome c; 8+ for lysozyme and 5+ for RNaseA at pH = 7; 6+ for insulin; 1+ for insulin A chain and 5+ for insulin B chain at pH = 3). The systems were placed in a cubic box, solvated with explicit simple point charge (SPC) model water molecules.37 The counter ions (chloride at pH 7; formate at pH 3) were added as necessary to ensure system neutrality, and geometry was optimized using 1000 steps of the steepest descent algorithm. For each system, a 1-ns MD simulation was performed at the isothermal isobaric (NPT; N, moles; P, pressure; T, temperature) ensemble. Temperature was kept at 300 K using the Velocity–rescale thermostat, 38 and pressure was maintained at 1 bar via the Parrinello–Rahman method.39 Periodic boundary conditions were used with a 1.4-nm cutoff for nonbonded interactions. Longrange electrostatic corrections were taken into account by the particle mesh Ewald method.40 The overall simulation window was 100 ns. RMSD, radius of gyration was monitored throughout all simulations. 3. Results and Discussions Figure 1 depicts a general workflow for the structure analyses of protein mixture. The basic theory of using MCE for peptide structure analyses has been reported in detail in our previous publication. 27 In this work, MCE experiments were carried out first for protein mixture. In MCE experiments as shown in Figure 1a, a constant liquid flow was generated by the application of a constant driving pressure, and a high DC voltage was applied to generate the electric field for protein separation. Precise flow rate of the running buffer (vr) was measured and calibrated using a neutral marker.41 Then hydrodynamic radius (Rh) of a protein could be calculated from its elution time using



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Equation 1.

(

𝑦 = 𝑣𝑟 +

q𝑈 ξL

)𝑡 +

ξ

q𝑈m

L ξ2(ⅇ

― m𝑡

Equation (1)

―1)

𝐿

𝑣𝑟 = 𝑡𝑀 ; ξ = 6𝜋𝜂𝑅ℎ in which q is the amount of charge possessed by the pKA, U is the DC potential applied across the separation capillary, L is total length of the separation capillary, t is migration time of sample, tM is migration time of neutral marker, η is the viscosity coefficient of the solution, m is the molar mass of sample.

Figure 1. The workflow of MCE restrained protein structure modelling method. (a) MCE experiments for protein mixture separation, and ion elution time was used for protein ellipsoid shape calculation. (b) The use of MCE experimental results restrains MD simulations for protein structure analyses. An ellipsoid shape approximation of proteins was then performed to obtain a function relationship between the ellipsoid aspect ratio 𝜙 = 𝑎/𝑐, and the ellipsoid short semi-axis length c through Equation 2:

𝑅𝐺 = 𝑅ℎ ― r𝑠 =

{

8𝑐 3

8𝑐 3

1

× ―

×

2𝜙 𝜙2 ― 1

+

2𝜙2 ― 1

3Ln(

𝜙 + 𝜙2 ― 1

, 𝜙>1 )

𝜙 ― 𝜙2 ― 1

(𝜙2 ― 1)2 1 2𝜙 ― 𝜙2 + 1

+

2(1 ― 2𝜙2)

3 ArcTan( 2 (1 ― 𝜙 )2

1 ― 𝜙2 𝜙

,𝜙

Mobility Capillary Electrophoresis Restrained Modelling Method for

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