Detection of membrane protein-therapeutic agent complexes by mass spectrometry
| Title: | Detection of membrane protein-therapeutic agent complexes by mass spectrometry |
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| Patent Number: | 9,536,718 |
| Publication Date: | January 03, 2017 |
| Appl. No: | 14/126650 |
| Application Filed: | June 18, 2012 |
| Abstract: | According to the present invention, there is provided a method of detecting a complex comprising a membrane protein bound to a therapeutic agent by mass spectrometry. The method comprises: (a) providing a solution comprising a detergent micelle in which said complex is contained; (b) providing a mass spectrometer comprising a nanoelectrospray ionization source, a mass analyzer and a detector; (c) vaporizing the solution using the nanoelectrospray ionization source under conditions such that the complex is released from the micelle; (d) ionizing the complex; (e) resolving the ionized complex using the mass analyzer; and (f) detecting the resolved complex using the detector. Also provided is a solution comprising a detergent micelle in which a complex is contained, wherein the complex comprises a membrane protein bound to a therapeutic agent. |
| Inventors: | Robinson, Carol V. (Oxford, GB); Wang, Sheila (Oxford, GB); Barrera, Nelson P. (Oxford, GB) |
| Assignees: | Oxford University Innovation Limited (Oxford, GB) |
| Claim: | 1. A method of detecting a complex comprising a membrane protein bound to a therapeutic agent by mass spectrometry, wherein the method comprises: (a) providing a solution comprising a detergent micelle in which said complex is contained, 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, and wherein the solution comprises a mass spectrometry compatible buffer; (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 complex is released from the micelle; (d) ionising the complex; (e) resolving the ionised complex using the mass analyser; and (f) detecting the resolved complex using the detector, 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 80 to about 240 V; (iii) the trap collision energy is from about 80 to about 240 V; (iv) the source temperature is from about 0 to about 50° C.; (v) the bias voltage is from about 40 to about 200 V; and (vi) the backing pressure is from about 1 to about 8 mBar. |
| Claim: | 2. A method according to claim 1 , wherein the membrane protein is an integral membrane protein. |
| Claim: | 3. A method according to claim 2 , wherein the integral membrane protein is a G protein-coupled receptor, a membrane transporter, an ATP-binding cassette transporter or a proton driven transporter. |
| Claim: | 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 and KirBac3.1. |
| Claim: | 5. A method according to claim 1 , wherein the therapeutic agent is a drug. |
| Claim: | 6. A method according to claim 1 , wherein the therapeutic agent is a non-polymeric organic compound having a molecular weight of less than 1000 Daltons. |
| Claim: | 7. A method according to claim 1 , wherein the therapeutic agent is an inhibitor or an activator. |
| Claim: | 8. A method according to claim 7 , wherein the therapeutic agent is a cyclic peptide inhibitor. |
| Claim: | 9. A method according to claim 1 , wherein the complex further comprises a lipid and/or a nucleotide. |
| Claim: | 10. A method according to claim 1 , wherein the solution comprises a non-ionic detergent. |
| Claim: | 11. A method according to claim 10 , wherein the solution comprises n-dodecyl-β-D-maltoside and/or nonylglucoside. |
| Claim: | 12. A method according to claim 1 , wherein the solution is an aqueous solution. |
| Claim: | 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. |
| Claim: | 14. A method according to claim 1 , wherein the solution is sprayed using a gold-coated nanoflow capillary. |
| Claim: | 15. A method according to claim 1 , wherein the complex is released from the micelle substantially intact. |
| Claim: | 16. A method according to claim 1 , wherein the mass spectrometer comprises a collision cell in which release and/or ionisation of the complex takes place. |
| Claim: | 17. A method according to claim 1 , wherein the mass spectrometer is operated with a bias voltage of from about 40 to about 200 V. |
| Claim: | 18. A method according to claim 1 , wherein the complex is detected by ion mobility-mass spectrometry. |
| Claim: | 19. A method according to claim 1 , wherein the structure or conformation of the complex is characterised. |
| Claim: | 20. A method according to claim 1 , wherein the mass spectrometer is operated with a bias voltage of from about 60 to about 180 V. |
| Claim: | 21. A method according to claim 1 , wherein the mass spectrometer is operated with a bias voltage of from about 80 to about 160 V. |
| Patent References Cited: | 6287857 September 2001 O'Riordan et al. 6790632 September 2004 Zweig 7288368 October 2007 Zweig 7575763 August 2009 Sligar et al. 7592008 September 2009 Sligar et al. 7595155 September 2009 Murakami 7691414 April 2010 Sligar et al. 2002/0182717 December 2002 Karlsson 2003/0219731 November 2003 Weinberger et al. 2004/0033624 February 2004 Zweig 2004/0259161 December 2004 Nilsson 2005/0196791 September 2005 Koopman et al. 2006/0019279 January 2006 Bosse et al. 2007/0026383 February 2007 Trubetskoy et al. 2007/0077291 April 2007 Petrenko 2007/0249060 October 2007 Kirschner et al. 2009/0117670 May 2009 Van Der Wijk et al. 2009/0275066 November 2009 Popot et al. 2010/0273677 October 2010 Lund-Johansen |
| Other References: | Laganowsky et al. “Mass spectrometry of intact membrane protein complexes”, Nature Protocols, 2013, v. 8, No. 4, pp. 639-651. cited by examiner Hernandez & Robinson, Determining the stoichiometry and interactions of macromolecular assemblies from mass spectrometry, Nature Protocols, 2007, 715-726, vol. 2, No. 3. cited by applicant Ho et al., Electrospray Ionisation Mass Spectrometry: Principles and Clinical Applications, Clin. Biochem. Rev., 2003, p. 3-12, vol. 24. cited by applicant Sharon et al., Evidence for Micellar Structure in the Gas Phase, J. Am. Chem. Soc., 2007, 8740-8746, vol. 129, No. 28. cited by applicant Chang et al., Structure of P-glycoprotein, Drug Metabolism Reviews: 9th Int'l Meeting of the Int'l Society for the Study of Xenobiotics (ISSX), 2010, 6-7, vol. 42, No. Suppl. 1. cited by applicant Ilag, L.L., et al., Drug Binding Revealed by Tandem Mass Spectrometry of a Protein-Micelle Complex: supportive information, Journal of the American Chemical Society, Nov. 1, 2004, vol. 126, No. 44, pp. 14362-14363. cited by applicant Barrera, N.P., et al., Mass spectrometry of membrane transporters reveals subunit stoichiometry and interactions, Nature Methods, Jul. 5, 2009, vol. 6, No. 8, pp. 585-587; http://www.nature.com/nmeth/journal/v6/n8/full/nmeth.1347.html#online-methods; & supplementary information. cited by applicant Barrera, N.P., et al., Micelles Protect Membrane Complexes from Solution to Vacuum, Science, Jul. 11, 2008, vol. 321, No. 5886, pp. 243-246. cited by applicant Wang, S.C., et al., Ion Mobility Mass Spectrometry of Two Tetrameric Membrane Protein Complexes Reveals Compact Structures and Differences in Stability and Packing, Journal of the American Chemical Society, Nov. 10, 2010, vol. 132, No. 44, pp. 15468-15470. cited by applicant Barrera, N.P., et al., Advances in the Mass Spectrometry of Membrane Proteins: From Individual Proteins to Intact Complexes, Annual Review of Biochemistry, Jul. 7, 2011, vol. 80, No. 1, pp. 247-271. cited by applicant Rakic-Martinez, et al., Listeria monocytogenes Strains Selected on Ciprofloxacin or the Disinfectant Benzalkonium Chloride Exhibit Reduced Susceptibility to Ciprofloxacin, Gentamicin, Benzalkonium Chloride, and Other Toxic Compounds, Applied and Environmental Microbiology, vol. 77, No. 24, pp. 8714-8721 Dec. 1, 2011. cited by applicant |
| Primary Examiner: | Gakh, Yelena G |
| Attorney, Agent or Firm: | Hodgson Russ LLP |
| Accession Number: | edspgr.09536718 |
| Database: | USPTO Patent Grants |
| Language: | English |
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