Detergents and methods
OwnerLeonhard Hagen URNER, Kevin PAGEL +5inventors
InventorsLeonhard Hagen URNER, Kevin PAGEL, Rainer HAAG, Dennis SHUTIN, Mark AGASID, Francesco FIORENTINO, Carol V. ROBINSON
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Abstract
A method of detecting a protein by mass spectrometry comprises: providing a solution comprising a hybrid detergent and a protein; providing a mass spectrometer comprising a nanoelectrospray ionisation source; vaporising the solution; ionising the protein; resolving the ionised protein; and detecting the resolved protein. The mass spectrometry methods may be used to interrogate the lipidome of a protein of interest, and/or for analysing membrane proteins in the form of complexes with ligands, and in particular lipids.
Claims25
A method of detecting a protein by mass spectrometry, wherein the method comprises:
- (a) providing a solution comprising a hybrid detergent and a protein;
- (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;
- (d) ionising the protein;
- (e) resolving the ionised protein using the mass analyser;
- and
- (f) detecting the resolved protein using the detector;
- wherein the hybrid detergent comprises a hybrid head group linked to a hydrophobic tail, wherein the hybrid head group comprises a first hydrophilic group and a second hydrophilic group which is different from the first hydrophilic head group, wherein each of the first and second hydrophilic groups is derived from a polyol or contains a charged group.
The method of any preceding claim, wherein the hybrid detergent has the formula:
- where:
- H represents the hybrid head group;
- L represents a linking group;
- and
- T represents the hydrophobic tail.
The method of any preceding claim, wherein the hybrid head group has the structure:
- where:
- A represents a trivalent group;
- H1 represents the first hydrophilic group;
- and
- H2 represents the second hydrophilic group,
- and preferably wherein the hybrid head group has the structure:
The method of any preceding claim, wherein the hybrid head group is joined to the hydrophobic tail by a linking group, L, selected from:
- hydrocarbylene, heterocyclylene, O, S, NR′, NR′—O, C(O)NR′, OC(O)NR′, OC(O)O, NR′C(O)NR′, NR′C(S)NR′, C(NR′)NR′, C(O), C(O)O, S(O)2, S(O), S(O)2O, S—S, CR′═N, CR′═N—NR′, C═N—NR′C(O), and combinations of up to three of these groups, where each R′ is independently selected from H, C1-4 alkyl and C1-4 alkoxy;
- and preferably wherein the linking group is selected from:
- O, S, C(O)O, O—C1-4 alkylene-aryl and OC(═O)NR′.
The method of any preceding claim, wherein the hydrophobic tail comprises a C6-100 alkyl group in which one or more methylene groups may be independently replaced by a unit selected from:
- C2-6 alkenylene, C2-6 alkynylene, arylene, O, S, NR″, NR″—O, C(O)NR″, OC(O)NR″, OC(O)O, NR″C(O)NR″, NR″C(S)NR″, C(NR″)NR″, C(O), S(O)2, S(O), S(O)2O, S—S, CR″═N, CR″═N—NR″, C═N—NR″C(O), where each R″ is independently selected from H, C1-4 alkyl and C1-4 alkoxy.
The method of any preceding claim, wherein the hybrid detergent has:
- a total number of carbons of up to 60, preferably up to 50, and more preferably up to 40;
- and/or
- a molecular weight of up to 1,000 Da, preferably up to 800 Da, and more preferably up to 650 Da.
The method of any preceding claim, wherein the protein is detected in the form of a complex with a lipid, such as a phospholipid or a lipopolysaccharide.
The method of any preceding claim, wherein the hybrid detergent is present in the solution at a concentration which is greater than or equal to the critical aggregation concentration (CAC) of the hybrid detergent in said solution, and preferably wherein:
- the hybrid detergent is present in the solution at a concentration which is from 1.5 to 3 times the CAC, and more preferably from 1.75 to 2.5 times the CAC;
- or
- the hybrid detergent is present in the solution at a concentration which is from 35 to 65 times the CAC, and more preferably from 45 to 55 times the CAC.
The method of any preceding claim, wherein the protein is detected substantially intact.
The method of any preceding claim, wherein the structure or conformation of the protein is characterised.
The method of any preceding claim, wherein the protein is a membrane protein.
A method of interrogating the lipidome of a protein of interest, said method comprising:
- providing at least three solutions comprising a detergent and the protein, a different detergent being used in each solution;
- providing a mass spectrometer comprising a nanoelectrospray ionisation source, a mass analyser and a detector, and for each of the solutions:
- vaporising the solution using the nanoelectrospray ionisation source;
- ionising the protein;
- resolving the ionised protein using the mass analyser;
- detecting the resolved protein using the detector;
- and
- determining the degree of lipidation in the detected protein;
- calculating at least one of the hydrophobic-hydrophilic balance (HLB) and the packing parameter (p value) of each of the detergents;
- and
- correlating the HLB and/or p value of the detergents with the degree of lipidation in the detected protein.
Dependencies are inferred from the claim text.