Analysis of polymeric protein and protein products

Claim 1

A method of analyzing a polymeric protein or polymeric protein product for the presence or absence of a ragged end at its C-or N-terminus, said method comprising the steps of: (a) digesting chemically or enzymatically said polymeric protein or polymeric protein product to produce N- or C-terminus fragments which are differentiable from non-terminal fragments of te digested protein or protein product; (b) subjecting the N- and/or C-terminus fragments obtained in step (a) to fast atom bombardment and high-mass mass spectrometry; and (c) analyzing the mass spectrometric data obtained and checking for the presence of fragments having a mass which would be unexpected from a study of a known complete molecule of the protein or protein product which is believed to be present, whereby the presence of such unexpected fragments indicates the presence of said ragged end.

Claim 2

A method of analyzing a polymeric protein or polymeric protein product for the presence or absence of a ragged end at its C-or N-terminus, said method comprising the steps of: (a) digesting chemically or enzymatically said polymeric protein or polymeric protein product to produce N- or C-terminus fragments which are differentiable from non-terminal fragments of te digested protein or protein product; (b) subjecting the N- and/or C-terminus fragments obtained in step (a) to fast atom bombardment and high-mass mass spectrometry; and (c) analyzing the mass spectrometric data obtained and checking for the presence of fragments having a mass which would be unexpected from a study of a known complete molecule of the protein or protein product which is believed to be present, whereby the presence of such unexpected fragments indicates the presence of said ragged end.

Claim 12

A method for locating the N- or C-terminal peptide in a polymeric protein or protein product, said method comprising the steps of: (a) digesting chemically or enzymatically said polymeric protein or polymeric protein product to produce N- or C-terminus fragments which are differentiable from non-terminal fragments, of the digested protein or protein product; (b) subjecting the protein fragments obtained in step (a) to fast atom bombardment and high-mass mass specrometry; (c) subjecting the products obtained in step (a) to a second digestion step, said second digestion step being different from said first chemical or enzymatic digestion; (d) subjecting the protein fragments of step (c) to fast atom bombardment and high-mass mass spectrometry; and (e) analyzing and comparing the mass spectrometric data obtained from both mass spectrometric exercises of steps (b) and (d) to establish whether or not some or all of the mass spectrometric signal peaks have shifted, and deducing from this the location of the terminal peptide.

Claim 13

A method for locating the N- or C-terminal peptide in a polymeric protein or protein product, said method comprising the steps of: (a) digesting chemically or enzymatically said polymeric protein or polymeric protein product to produce N- or C-terminus fragments which are differentiable from non-terminal fragments, of the digested protein or protein product; (b) subjecting the protein fragments obtained in step (a) to fast atom bombardment and high-mass mass specrometry; (c) subjecting the products obtained in step (a) to a second digestion step, said second digestion step being different from said first chemical or enzymatic digestion; (d) subjecting the protein fragments of step (c) to fast atom bombardment and high-mass mass spectrometry; and (e) analyzing and comparing the mass spectrometric data obtained from both mass spectrometric exercises of steps (b) and (d) to establish whether or not some or all of the mass spectrometric signal peaks have shifted, and deducing from this the location of the terminal peptide.

Claim 16

A method of assigning disulphide bridges in polymeric protein or polymeric protein products, said method comprising the steps of: (a) digesting said protein or protein product in an unreduced state; (b) subjecting the digest mixture or, one or more fractionated components thereof to fast atom bombardment and high-mass mass spectrometry; (c) treating the digest mixture or, one or more fractionated components thereof to a molecular-weight altering step; and (d) performing mass spectrometric analysis upon the products obtained in step (c) and comparing the mass spectrometric results to identify disulphide bridged peptides by their characteristic masses.

Claim 17

A method of assigning disulphide bridges in polymeric protein or polymeric protein products, said method comprising the steps of: (a) digesting said protein or protein product in an unreduced state; (b) subjecting the digest mixture or, one or more fractionated components thereof to fast atom bombardment and high-mass mass spectrometry; (c) treating the digest mixture or, one or more fractionated components thereof to a molecular-weight altering step; and (d) performing mass spectrometric analysis upon the products obtained in step (c) and comparing the mass spectrometric results to identify disulphide bridged peptides by their characteristic masses.