Patent ID: 7163803

Claim:
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 R 2 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.