DETECTION OF MEMBRANE PROTEINS
According to the present invention, there is provided a method of detecting a membrane protein by mass spectrometry, the method comprising the steps of: (a) providing a solution comprising a detergent micelle in which said membrane protein is contained, wherein said solution contains a polyoxyalkylene glycol detergent; (b) providing a mass spectrometer comprising a nanoelectrospray ionisation source, a mass analyser and a detector; (c) vaporising the solution using the nanoelectrospray ionisation source under conditions such that the membrane protein is released from the micelle; (d) ionising the membrane protein; (e) resolving the ionised membrane protein using the mass analyser; and (f) detecting the resolved membrane protein using the detector. Also provided are reagents for use in said method.
1 . A method of detecting a membrane protein by mass spectrometry, wherein the method comprises:
(a) providing a solution comprising a detergent micelle in which said membrane protein is contained, wherein said solution contains a polyoxyalkylene glycol detergent;
(b) providing a mass spectrometer comprising a nanoelectrospray ionisation source, a mass analyser and a detector;
(c) vaporising the solution using the nanoelectrospray ionisation source under conditions such that the membrane protein is released from the micelle;
(d) ionising the membrane protein;
(e) resolving the ionised membrane protein using the mass analyser; and
(f) detecting the resolved membrane protein using the detector.
2 . A method according to claim 1 , wherein the membrane protein is an integral membrane protein.
3 . A method according to claim 2 , wherein the integral membrane protein is a G protein-coupled receptor, a membrane transporter, a drug efflux pump, an ATP-binding cassette transporter or a proton driven transporter.
4 . A method according to claim 1 , wherein the membrane protein is selected from EmrE, LmrP, MscL, BtuCD, BtuC 2 D 2 , LmrCD, MacB, MexB, P-gp, MsbA, NorM, KirBac3.1, AmtB and Aquaporin Z.
5 . A method according to any preceding claim 1 , wherein the membrane protein is in the form of a complex with one or more ligands.
6 . A method according to claim 5 , wherein the one or more ligands are selected from therapeutic agents, lipids and nucleosides.
7 . A method according to claim 5 , wherein the membrane protein is in the form of a complex with a therapeutic agent, e.g. a drug, e.g. a non-polymeric organic compound having a molecular weight of less than 1000 Daltons.
8 . (canceled)
9 . A method according to claim 5 , wherein the one or more ligands include one or more lipids, e.g. one or more phospholipids.
10 . (canceled)
11 . A method according to claim 1 , wherein the detergent is present in the solution at a concentration which is greater than or equal to the critical micelle concentration of the detergent in said solution.
12 . A method according to claim 1 , wherein the solution is an aqueous solution.
13 . A method according to claim 1 , wherein the molar ratio of the detergent to the membrane protein in the solution is from about 10:1 to about 150:1.
14 . (canceled)
15 . A method according to claim 1 , wherein the membrane protein is released from the micelle substantially intact.
16 . A method according to claim 1 , wherein the mass spectrometer comprises a collision cell in which release and/or ionisation of the membrane protein takes place.
17 . A method according to claim 1 , wherein the mass spectrometer is operated under one or more of the following conditions: (i) the capillary voltage of the nanoelectrospray ionisation source is from about 0.8 to about 2.2 kV; (ii) the cone voltage of the nanoelectrospray ionisation source is from about 10 to about 240 V; (iii) the trap collision energy is from about 50 to about 240 V; (iv) the source temperature is from about 0 to about 50° C.; (v) the bias voltage is from about 20 to about 200 V; and (vi) the backing pressure is from about 1 to about 8 mBar.
18 . (canceled)
19 . A method according to claim 1 , wherein the membrane protein is detected by ion mobility-mass spectrometry.
20 . (canceled)
21 . A method according to claim 1 , wherein the mass spectrometer is operated at a laboratory frame energy of from about 500 to about 5000 electron volts, e.g. from about 500 to about 1500 electron volts.
22 . A method according to claim 1 , wherein the polyoxyalkylene glycol detergent has a molecular weight of at least 300 Da, e.g. from 300 Da to 1000 Da, e.g. from 300 Da to 600 Da or from 500 Da to 1000 Da.
23 . A method according to claim 1 , wherein the polyoxyalkylene glycol detergent is a polyoxyethylene glycol detergent, e.g. tetraethylene glycol monooctyl ether (“C 8 E 4 ”), pentaethylene glycol monooctyl ether (“C 8 E 5 ”), pentaethylene glycol monodecyl ether (“C 10 E 5 ”), polyoxyethylene (8) dodecyl ether (“C 12 E 8 ”), polyoxyethylene (20) cetyl ether (“C 16 E 20 ”) or polyethylene glycol tert-octylphenyl ether (t-oct-C 6 H 4 —(OCH 2 CH 2 ) x OH, where x is 9-10).
24 . (canceled)
25 . A method according to claim 1 , wherein the solution contains a plurality of polyoxyalkylene glycol detergents.
26 . A solution comprising a detergent micelle in which a complex is contained, wherein the complex comprises a membrane protein bound to one or more ligands, and wherein the solution comprises a polyoxyalkylene glycol detergent.
27 - 30 . (canceled)