Method for characterizing polypeptides
AssigneeElectrophoretics Limited
InventorsChristian Hamon, Andrew Thompson, Thomas Neumann, Robert Johnstone, Abdul Karim Abed Mohammed
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Abstract
Provided is a method for characterizing a polypeptide, which method comprises the steps of: (a) optionally reducing cysteine disulphide bridges in the polypeptide to form free thiols, and capping the free thiols; (b) cleaving the polypeptide with a sequence specific cleavage reagent to form peptide fragments; (c) optionally deactivating the cleavage reagent; (d) capping one or more ε-amino groups that are present with a lysine reactive agent; (e) analyzing peptide fragments by mass spectrometry to form a mass fingerprint for the polypeptide; and (f) determining the identity of the polypeptide from the mass fingerprint.
Claims22
A method for determining the identity of a polypeptide, which method comprises the steps of:
- (a) optionally reducing cysteine disulphide bridges in the polypeptide to form free thiols, and capping the free thiols;
- (b) cleaving the polypeptide with a sequence specific cleavage reagent to form peptide fragments;
- (c) optionally deactivating the cleavage reagent;
- (d) capping one or more ε-amino groups that are present with a lysine reactive agent, wherein the lysine reactive agent comprises a hindered Michael reagent;
- (e) analyzing peptide fragments by mass spectrometry to determine a peptide mass fingerprint for the polypeptide;
- and
- (f) determining the identity of the polypeptide from the peptide mass fingerprints;
- wherein the hindered Michael agent comprises a compound having the following structure:
- wherein X is an electron withdrawing group that stabilizes a negative charge;
- the R groups independently comprise a hydrogen, a halogen, an alkyl, an aryl, or an aromatic group with the proviso that at least one of the R groups comprises a sterically hindering group;
- and the group R2 comprises a hydrogen, a halogen, a hydrocarbon group, an electron withdrawing group and/or a linker that attaches to an affinity capture functionality or a solid phase support.
A method for determining the identity of one or more polypeptides in a sample comprising a population of polypeptides, which method comprises the steps of:
- (a) optionally reducing cysteine disulphide bridges in one or more of the polypeptides to form free thiols, and capping the free thiols;
- (b) separating one or more of the polypeptides from the sample;
- (c) cleaving the one or more polypeptides with a sequence specific cleavage reagent to form peptide fragments;
- (d) optionally deactivating the cleavage reagent;
- (e) capping one or more ε-amino groups that are present with a lysine reactive agent, wherein the lysine reactive agent comprises a hindered Michael reagent;
- (f) analyzing the peptide fragments by mass spectrometry to form a peptide mass fingerprint for the one or more polypeptides;
- and
- (g) determining the identity of the one or more polypeptides from the peptide mass fingerprint;
- wherein the hindered Michael agent comprises a compound having the following structure:
- wherein X is an electron withdrawing group that stabilizes a negative charge;
- the R groups independently comprise a hydrogen a halogen, an alkyl, an aryl, or an aromatic group with the proviso that at least one of the R groups comprises a sterically hindering group;
- and the group R2 comprises a hydrogen, a halogen, a hydrocarbon group, an electron withdrawing group and/or a linker that attaches to an affinity capture functionality or a solid phase support.
A method for comparing peptide mass fingerprints for a plurality of samples, each sample comprising one or more polypeptides, which method comprises the steps of:
- (a) optionally reducing cysteine disulphide bridges and capping the free thiols in one or more polypeptides from each of the samples;
- (b) separating one or more polypeptides from each of the samples;
- (c) cleaving the one or more polypeptides with a sequence specific cleavage reagent to form peptide fragments;
- (d) optionally deactivating the cleavage reagent;
- (e) capping one or more ε-amino groups that are present with a lysine reactive agent, wherein the lysine reactive agent comprises a hindered Michael reagent;
- (f) analyzing the peptide fragments by mass spectrometry to form a peptide mass fingerprints for the one or more polypeptides;
- and
- (g) determining the identity of the one or more polypeptides from the peptide mass fingerprints;
- and
- (h) comparing the peptide mass fingerprints of the plurality of samples;
- wherein the hindered Michael agent comprises a compound having the following structure:
- wherein X is an electron withdrawing group that stabilizes a negative charge;
- the R groups independently comprise a hydrogen, a halogen, an alkyl an aryl, or an aromatic group with the proviso that at least one of the R groups comprises a sterically hindering group;
- and the group R2 comprises a hydrogen a halogen, a hydrocarbon group, an electron withdrawing group and/or a linker that attaches to an affinity capture functionality or a solid phase support.
Dependencies are inferred from the claim text.