A method for analysing a membrane protein
OwnerCarol ROBINSON, Corinne LUTOMSKI +4inventors
InventorsCarol ROBINSON, Corinne LUTOMSKI, Tarick EL-BABA, Idlir LIKO, John SYKA, Josh HINKLE
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Abstract
The disclosure concerns a method of analysing a sample comprising an intact membrane protein, in a sample comprising non-synthetic biological membrane material. The method involves irradiating a gas phase proto membrane protein ion with infrared irradiation from a laser source.
Claims29
A method of analysing a sample comprising an intact membrane protein, the method comprising:
- (i) providing a sample comprising a non-synthetic biological membrane material comprising the intact membrane protein, a solvent, and bulk lipid molecules;
- (ii) ionising the sample to produce a gas phase proto membrane protein ion, said gas phase proto membrane protein ion comprising the intact membrane protein non-specifically bound to one or more bulk lipid molecules and to one or more residual solvent molecules;
- (iii) confining the gas phase proto membrane protein ion;
- (iv) detaching the one or more residual solvent molecules and one or more non-specifically bound bulk lipid molecules from the intact membrane protein by irradiating the confined gas phase proto membrane protein ion with infrared radiation from a laser source to produce a gas phase intact membrane protein ion;
- and
- (v) m/z analysing and detecting the gas phase intact membrane protein ion and/or an ion derived therefrom.
The method of any preceding claim wherein, in the non-synthetic biological membrane material provided in step (i), the intact membrane protein is present in or attached to a lipid bilayer comprising the bulk lipid molecules.
The method of any preceding claim wherein step (i) comprises sonicating the sample.
The method of any preceding claim wherein the sample is ionised by electrospray ionisation, preferably by nanoelectrospray ionisation or by static electrospray ionisation.
The method of any preceding claim wherein the sample is ionised by native electrospray ionisation.
The method of any preceding claim wherein the gas phase proto membrane protein ion of step (ii) comprises one or more submicron-scale membrane fragments or vesicles associated with the intact membrane protein and with solvent molecules.
The method of any preceding claim which additionally comprises exposing the gas phase proto membrane protein ion to a collision gas to remove one or more residual solvent molecules by collision-induced dissociation.
The method of any preceding claim wherein a wavelength of the infrared radiation from the laser source preferentially excites one or more vibrational modes of one or more bonds within the non-specifically bound bulk lipid molecules.
The method of any preceding claim wherein the infrared radiation from the laser is continuous wave radiation.
The method of any preceding claim wherein the wavelength of the infrared radiation from the laser source is from 780 nm to 1 mm, preferably from 1.4 μm to 1 mm, more preferably from 3 μm to 100 μm, most preferably between 9 and 11 μm.
The method of any preceding claim, wherein the wavelength of the infrared radiation from the laser source has a principal wavelength at one or more of 10.6 μm, 10.2 μm, 9.6 μm, and 9.3 μm.
The method of any preceding claim wherein the irradiation with the infrared radiation is performed for a period of up to 1 second, preferably for a period of less than 50 ms.
The method of any preceding claim wherein step (v) comprises:
- (va) subjecting the gas phase intact membrane protein ion to an ion transformative process to produce a first-generation product ion;
- and
- (vb) m/z selecting the first-generation product ion.
The method according to any preceding claim wherein the gas phase proto membrane protein ion and/or the gas phase intact membrane protein ion is held in an ion trap or transmitted through an ion guide or mass filter, and the method comprises:
- m/z isolating the gas phase proto membrane protein ion and/or the gas phase intact membrane protein ion by ejecting ions of m/z outside one or more pre-determined m/z ranges from the ion trap, ion guide or mass filter.
The method according to any preceding claim which comprises, prior to any step wherein an ion is detected, or subjected to an ion transformative process, reducing the charge of a precursor ion to produce the said ion.
The method according to any preceding claim which comprises determining irradiation parameters to be used in step (iii) by repeatedly performing the process and varying the energy of the laser radiation and/or the period during which the irradiation is performed until the gas phase intact membrane protein ion can be detected after step (iv).
The method according to any preceding claim wherein the intact membrane protein comprises two or more non-covalently associated monomers, which monomers may be the same or different.
The method according to any preceding claim, wherein the gas phase intact membrane protein ion comprises one or more non-covalently bound ligands.
The method according to any preceding claim wherein the sample comprises a mass-spectrometry compatible buffer, which preferably maintains the structural integrity of the intact membrane protein in a submicron-scale membrane fragment or vesicle associated with the intact membrane protein and solvent molecules.
The method according to any preceding claim which is a mass spectrometric method, wherein step (v) comprises determining the m/z ratio of said gas phase intact membrane protein ion and/or an ion derived therefrom.
The method according to any preceding claim wherein the sample, the non-synthetic biological membrane material and/or the gas phase proto membrane protein ion is at least substantially free of detergent.
Dependencies are inferred from the claim text.