CN113433196A

CN113433196A - Non-denaturing mass spectrometry of membrane protein complexes using nonaethylene glycol monododecyl ether

Info

Publication number: CN113433196A
Application number: CN202010201279.5A
Authority: CN
Inventors: 周敏; 卢颖洪; 陈沐
Original assignee: Nanjing University of Science and Technology
Current assignee: Nanjing University of Science and Technology
Priority date: 2020-03-20
Filing date: 2020-03-20
Publication date: 2021-09-24
Anticipated expiration: 2040-03-20
Also published as: CN113433196B

Abstract

The invention discloses a non-denaturing mass spectrometry analysis method for a membrane protein complex by using nonaethylene glycol monododecyl ether. The method comprises the steps of firstly carrying out ultrafiltration desalination on a purified membrane protein complex in the presence of C12E9, then replacing the purified membrane protein complex into an ammonium acetate buffer solution containing C12E9, further removing a surfactant in an original solution by dialysis with the ammonium acetate buffer solution containing C12E9, and finally carrying out mass spectrometry on the membrane protein complex containing C12E9 and a protein standard product respectively under the same mass spectrometry condition. The invention adopts C12E9 to replace the traditional surfactant DDM, greatly reduces the energy state of the membrane protein, and effectively maintains the natural conformation of the membrane protein, thereby being capable of quickly and accurately providing the information of the structural biology of the membrane protein, and the effect is obviously better than the traditional surfactant DDM.

Description

Non-denaturing mass spectrometry of membrane protein complexes using nonaethylene glycol monododecyl ether

Technical Field

The invention belongs to the technical field of protein structure biology, and relates to a non-denaturing mass spectrometry analysis method for a membrane protein complex by using nonaethylene glycol monododecyl ether (C12E 9).

Background

Membrane proteins are responsible for a wide range of biological functions, and once they fail, they often cause serious diseases such as cancer and the like. Although membrane proteins account for only one third of the human genome, more than half of the drug targets are membrane proteins. As an important subject of cancer and disease, membrane proteins have been studied for their structure by conventional structural biological means such as X-ray crystallography and nuclear magnetic resonance. However, the difficulty of the structural biological research of the membrane protein is often caused by the difficulties of the expression amount and solubility of the membrane protein. Furthermore, for conventional X-ray crystallography, the resolution of the crystal structure is not sufficient to resolve the binding domains.

BtuCD is a class of ABC transporters, and the physiological function of BtuCD is to use the energy of hydrolyzing ATP for coupling transmembrane transfer of a substrate. The BtuCD comprises two Nucleotide Binding Domains (NBD) for hydrolyzing ATP, and the energy of the hydrolyzed ATP is utilized to control the structural change of a transmembrane domain, so that the transport process of the vitamin B12 is realized. Coli BtuCD is a type II ABC transporter that is structurally and mechanistically similar to transferrin and heme transporters, and its function is associated with virulence of certain gram-negative pathogens. BtuCD membrane protein complex is often used as a model membrane protein complex for membrane protein research.

Non-denaturing mass spectrometry is a rapid and sensitive analytical method that can provide structural information of both the protein complex and the bound small molecule region. In general, to maintain the native conformation of the membrane protein, high concentrations of surfactant are added in large amounts to the solution, so that micelles of surfactant of varying sizes are formed. Due to such circumstances, the membrane protein was initially unavailable for mass spectrometric analysis until later studies found that the membrane protein encapsulated by n-Dodecyl β -D-maltoside (DDM) surfactant could be transferred into the gas phase as a whole. By bombardment with high energy particles, the surfactant molecules can be stripped from the surface of the membrane protein and the interactions between the various subunits of the membrane protein complex are maintained.

Membrane protein mass spectrometry first requires the use of nanoelectrospray to ionize and transfer a surfactant-containing purified membrane protein solution to a mass spectrometer. The main component of the protein spray obtained by the method of mass spectrum ionization is formed by wrapping a surfactant by a charged membrane protein complex. It is clear that in this case the molecular weight of the protein complex cannot be accurately measured. It is therefore necessary to remove the surfactant molecules by thermodynamic activation, this being achieved primarily by increasing the energy of the collision activation process, followed by screening out the target molecules by a quadrupole mass analyser. The accelerated ions pass through a collision cell filled with an inert gas, thereby dissociating the surfactant molecules from the protein complex, and thereby detaching intact protein complexes without damage. Through further screening of the radio frequency conductance, the membrane protein complex ions are transferred to a time-of-flight mass spectrometer by a thruster. Thus, the membrane protein complex can be finally detected by the microchannel plate detector.

The collisional activation process not only releases the membrane protein complex from the surfactant micelle, but the energy-increasing effect also includes dissociation of the membrane protein complex. Too high activation energy can also lead to the collapse of local structures, and when the energy crosses a certain threshold, the collapsed denatured protein subunits can even be ejected from the membrane protein complex. Due to asymmetric partitioning during charge separation of the parent complex, a certain protein subunit is loaded with too high a charge, while the remaining complex has only a small charge. The stripping facilitates high resolution analysis of complexes with less charge, and the results of stripping also facilitate stoichiometric studies of membrane protein subunits and compositional analysis of multimeric protein complexes.

In view of the above, the key to the investigation of the non-denaturing mass spectrum of membrane protein complexes is to find a suitable surfactant that must be able to maintain the native conformation of the membrane protein in aqueous solution on the one hand, and to facilitate its removal at lower energy states in mass spectrometry experiments on the other hand. Currently, non-denaturing mass spectrometry of membrane protein complexes is mainly performed using surfactant DDM, but DDM is expensive and the average charge state of the membrane protein complex in the presence of DDM is high, the membrane protein complex is unstable, and it is difficult to remove DDM at a lower energy state (Nat chem.2014Mar; 6(3):208-215.doi: 10.1038/nchem.1868.).

Disclosure of Invention

Aiming at the characteristics and difficulties of the non-denaturing mass spectrum of the membrane protein complex, the invention provides a non-denaturing mass spectrum analysis method of the membrane protein complex by using nonaethylene glycol monododecyl ether. The method adopts nonaethylene glycol monododecyl ether as a surfactant to perform non-denaturing mass spectrometry analysis on the membrane protein complex.

The technical scheme of the invention is as follows:

the method for performing non-denaturing mass spectrometry analysis on the membrane protein complex by using the nonaethylene glycol monododecyl ether comprises the following specific steps:

firstly, desalting the purified membrane protein complex by an ultrafiltration membrane in the presence of nonaethylene glycol monododecyl ether, replacing the desalted membrane protein complex into ammonium acetate buffer solution which contains the nonaethylene glycol monododecyl ether and has pH of 8.0, and replacing a protein standard product into the ammonium acetate buffer solution with pH of 7.4;

secondly, dialyzing the membrane protein compound solution obtained in the first step by adopting an ammonium acetate buffer solution containing the nonaethylene glycol monododecyl ether, and further removing a surfactant in the original solution to obtain a membrane protein compound solution containing the nonaethylene glycol monododecyl ether;

thirdly, respectively carrying out mass spectrum analysis on the membrane protein complex containing the nonaethylene glycol monododecyl ether in the solution and a protein standard substance under the same mass spectrum condition to obtain non-denaturing mass spectrum data of the membrane protein complex and the protein standard substance and collecting respective ion mobility data;

fourthly, processing the obtained ion mobility data to obtain the drift time distribution of the membrane protein compound and the protein standard substance;

and fifthly, obtaining the collision cross-sectional area of the membrane protein complex according to the drift time distribution and the theoretical collision cross-sectional area of the protein standard and the drift time distribution of the membrane protein complex.

In the first step, the membrane protein complex is known in the art, and may be a BtuCD membrane protein complex or other known membrane protein complexes such as ATPase complex, heme transporter complex, ferroportin complex, etc., or may be unknown membrane protein complexes.

In the first step, when the membrane protein complex is desalted, the temperature is 4 ℃, the centrifugal rotating speed is 14000rcf, the time is not less than 30min, and the process is repeated for not less than 5 times.

In the first step, the protein standard is alcohol dehydrogenase, canavalin, avidin, beta-lactoglobulin, myosin, cytochrome c, and serum amyloid.

In the third step, the ion mobility mass spectrometer conditions were: the needle point voltage is 1.5kV, the taper hole voltage is 10V, the source compensation voltage is 10V, the primary collision chamber voltage is 2V, the secondary collision chamber voltage is 2V, the counter voltage between the primary collision chamber and the ion mobility separation chamber is 28V, the argon flow rate in the collision chamber is 2mL/min, the helium flow rate in the helium chamber is 150mL/min, nitrogen is used as separation gas in the ion mobility separation chamber, the flow rate is 60mL/min, the traveling wave speed of the ion mobility separation chamber is 1100m/s, and the traveling wave amplitude of the ion mobility separation chamber is 37V.

Compared with the prior art, the invention has the following advantages:

the invention adopts the nonaethylene glycol monododecyl ether to replace the traditional surfactant DDM, can more effectively help the membrane protein maintain the natural conformation in the water solution, and keeps the low energy state in the mass spectrum experiment, is beneficial to the non-denaturing mass spectrum analysis of the membrane protein, and is obviously superior to the traditional surfactant DDM.

Drawings

Fig. 1 is the relative signal intensity of BtuCD membrane protein complexes under different energy states in the presence of DDM and C12E9, respectively.

Fig. 2 is the average charge state of BtuCD membrane protein complex in the presence of DDM and C12E 9.

FIG. 3 shows the CIU (collagen-induced unfolding) of BtuCD membrane protein complex in the presence of C12E9 and DDM, respectively.

Fig. 4 shows the existence state of BtuCD membrane protein complex in C12E9 under different collision voltages (collision voltage).

Detailed Description

The present invention will be described in further detail with reference to the following examples and the accompanying drawings.

Example 1

Firstly, desalting a group of BtuCD membrane protein complexes by using a 10kD ultrafiltration membrane in the presence of C12E 9; desalting another group of BtuCD membrane protein complexes by using a 10kD ultrafiltration membrane in the presence of DDM; all protein standards (including alcohol dehydrogenase, canavalin, avidin, beta-lactoglobulin, myosin, cytochrome c, and serum amyloid) were desalted directly with a 10kD ultrafiltration membrane; the desalting treatment conditions were: the centrifugation temperature is 4 ℃, the centrifugation rotating speed is 14000rcf, the centrifugation is carried out for 30min, and the centrifugation is repeated for 5 times. Then replacing BtuCD membrane protein complex subjected to ultrafiltration desalination in the presence of C12E9 into ammonium acetate buffer solution containing C12E9 and pH8.0, replacing BtuCD membrane protein complex subjected to ultrafiltration desalination in the presence of DDM into ammonium acetate buffer solution containing DDM and pH8.0, and replacing protein standard product subjected to ultrafiltration desalination into ammonium acetate buffer solution containing pH7.4;

secondly, the BtuCD membrane protein complex solution replaced in the ammonium acetate buffer solution containing C12E9 and pH8.0 is dialyzed by adopting the ammonium acetate buffer solution containing C12E9, the BtuCD membrane protein complex solution replaced in the ammonium acetate buffer solution containing DDM and pH8.0 is dialyzed by adopting the ammonium acetate buffer solution containing DDM, and the surfactant in the original solution is further removed, so that the BtuCD membrane protein complex solution containing C12E9 and the BtuCD membrane protein complex solution containing DDM are respectively obtained;

and thirdly, respectively sucking 2 mu L of BtuCD membrane protein complex solution with the final concentration of 10uM and containing C12E9, 2 mu L of BtuCD membrane protein complex solution with the final concentration of 10uM and containing DDM and 2 mu L of protein standard substance mass spectrum with the final concentration of 5uM, and adjusting instrument parameters such as: the working mode is an ion mobility mode, the ionization mode is a positive ion mode, the needle point voltage is 1.5kV, the taper hole voltage is 10V, the source compensation voltage is 10V, the primary collision chamber voltage is 2V, the secondary collision chamber voltage is 2V, the counter voltage between the primary collision chamber and the ion mobility separation chamber is 28V, the argon flow rate in the collision chamber is 2mL/min, the helium chamber gas flow rate is 150mL/min, nitrogen is used as separation gas in the ion mobility separation chamber, the flow rate is 60mL/min, the ion mobility separation chamber traveling wave speed is 1100m/s, the ion mobility separation chamber traveling wave amplitude is 37V, and the ion mobility data of a membrane protein compound BtuCD and each protein standard are collected;

fourthly, processing the ion mobility data obtained in the third step through Masslynx and Driftscope software to obtain the drift time distribution of the BtuCD membrane protein compound and each protein standard;

and fifthly, obtaining the collision cross-sectional area of the BtuCD membrane protein compound according to the drift time distribution and the theoretical collision cross-sectional area of the protein standard substance and the drift time distribution of the BtuCD membrane protein compound, and finally analyzing the BtuCD membrane protein compound according to the drift time distribution and the collision cross-sectional area.

TABLE 1 Mass Spectrometry results of BtuCD membrane protein complexes in the presence of DDM and C12E9

Table 1 shows the results of mass spectrometry analysis of BtuCD membrane protein complexes in the presence of DDM and C12E9, respectively, and from CCS results, it can be seen that BtuCD membrane protein complexes are in a lower energy state and have a more compact structure, closer to the natural state.

FIG. 1 shows the relative signal intensities of BtuCD membrane protein complexes in the presence of DDM and C12E9, respectively, at different energy states. It can be seen that a better signal can still be obtained at C12E9 under very low excitation conditions.

Fig. 2 is the average charge state of BtuCD membrane protein complex in the presence of DDM and C12E 9. Wherein the average charge state of the BtuCD membrane protein complex in the presence of DDM is 22.5, the average charge state of the BtuCD membrane protein complex in the presence of C12E9 is about 19.5, and the lower charge state indicates that the stability of the protein complex is enhanced, which indicates that the BtuCD membrane protein complex has a more stable structure in the presence of C12E 9.

FIG. 3 shows the CIU (collagen-induced unfolding) of BtuCD membrane protein complex in the presence of C12E9 and DDM, respectively. (a) The ion mobility mass spectrum of BtuCD membrane protein complex in C12E9, (b) the distribution of the complete membrane protein complex in (a) and various sub-complexes and monomers with energy change, and (C) the ion mobility mass spectrum of BtuCD membrane protein complex in DDM. It can be seen that the CCS (average cross-sectional area) of the protein complex is smaller in C12E9 and the stability is enhanced.

Fig. 4 shows the existence state of BtuCD membrane protein complex in C12E9 under different collision voltages (collision voltage). In this series of graphs, collision energy gradually increases from bottom to top. In each mass spectrum, the protein monomer is in the lower charge state and the intact protein complex is in the higher charge state. Due to the stabilizing effect of C12E9 on membrane protein complexes, intact complexes could be observed at lower energy states (Cone voltage 100V). With the increase of the collision voltage, the membrane protein sub-complex and the monomer of the membrane protein gradually appear on the mass spectrum.

Claims

1. The method for performing non-denaturing mass spectrometry analysis on the membrane protein complex by using the nonaethylene glycol monododecyl ether is characterized by comprising the following specific steps of:

2. The method of claim 1, wherein in the first step, the membrane protein complex is a BtuCD membrane protein complex, an ATPase complex, a heme transporter complex, an iron transporter complex, or an unknown membrane protein complex.

3. The non-denaturing mass spectrometry method of claim 1, wherein in the first step, the membrane protein complex is desalted at 4 ℃, the centrifugation speed is 14000rcf, the time is not less than 30min, and the repetition is not less than 5 times.

4. The method of claim 1, wherein in the first step, the protein standard is selected from the group consisting of alcohol dehydrogenase, concanavalin, avidin, beta-lactoglobulin, myosin, cytochrome c, and serum amyloid.

5. The method of non-denaturing mass spectrometry of claim 1, wherein in the third step, the ion mobility spectrometer conditions are: the needle point voltage is 1.5kV, the taper hole voltage is 10V, the source compensation voltage is 10V, the primary collision chamber voltage is 2V, the secondary collision chamber voltage is 2V, the counter voltage between the primary collision chamber and the ion mobility separation chamber is 28V, the argon flow rate in the collision chamber is 2mL/min, the helium flow rate in the helium chamber is 150mL/min, nitrogen is used as separation gas in the ion mobility separation chamber, the flow rate is 60mL/min, the traveling wave speed of the ion mobility separation chamber is 1100m/s, and the traveling wave amplitude of the ion mobility separation chamber is 37V.