Methods for the high-resolution identification of solvent-accessible amide hydrogens in polypeptides or proteins and for characterization of the fine structure of protein binding sites
AssigneeCarta Proteomics, Inc.
InventorVirgil L. Woods, Jr.
Life of this patent
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Abstract
The present invention provides methods whereby the positions of peptide amide groups that are labeled with a heavy hydrogen in a polypeptide or protein can be localized at high resolution. The methods are useful for determining which peptide amide groups in a polypeptide or protein are accessible to solvent, mapping the binding site and/or binding surface of a binding protein, and/or studying allosteric or other conformational changes in a polypeptide or protein which alter the rates at which certain peptide amide hydrogens exchange with solvent.
Claims32
A method of determining, at a resolution of about 1-5 amino acid residues, the position of a peptide amide group labeled with a heavy hydrogen in a protein of known amino acid sequence, said method comprising the steps of:
- (a) progressively degrading, under conditions of slow hydrogen exchange, said protein into at least one series of subfragments, wherein each subfragment in each of said series is shorter than the preceding subfragment in the series by about 1-5 amino acid residues at one terminus;
- and
- (b) quantifying the amount of heavy hydrogen label on each subfragment;
- and
- (c) correlating the amount of heavy hydrogen label of each subfragment with the amino acid sequence of the protein, thereby localizing the position of the labeled peptide amide group in the protein to within about 1-5 amino acid residues.
A method of determining, at a resolution of about 1-5 amino acid residues, the position of a peptide amide group labeled with a heavy hydrogen in a protein of known amino acid sequence, said method comprising the steps of:
- (a) fragmenting, under conditions of slow hydrogen exchange, the protein, into a plurality of fragments;
- (b) determining, under conditions of slow hydrogen exchange, which of said fragments are labeled with heavy hydrogen;
- (c) progressively degrading each fragment which is labeled with heavy hydrogen under conditions of slow hydrogen exchange to obtain at least one series of subfragments, wherein each subfragment in each of said series is shorter than the preceding subfragment in the series by about 1-5 amino acid residues at one terminus;
- (d) quantifying the amount of heavy hydrogen label on each subfragment;
- and
- (c) correlating the amount of heavy hydrogen label of each subfragment with the amino acid sequence of the labeled fragment, thereby localizing the position of the labeled peptide amide in the fragment to within about 1-5 amino acid residues.
A method of determining, at a resolution of about 1-5 amino acid residues, which peptide amide groups in a protein of known amino acid sequence are accessible to solvent, said method comprising the steps of:
- (a) contacting a protein with heavy hydrogen under conditions wherein peptide amide hydrogens of the protein which are freely accessible to solvent exchange with, and become selectively labeled by, heavy hydrogen;
- (b) progressively degrading the selectively labeled protein under conditions of slow hydrogen exchange into at least one series of subfragments, wherein each subfragment in each of said series is shorter than the preceding subfragment in the series by about 1-5 amino acid residues at one terminus;
- (c) quantifying the amount of heavy hydrogen label on each subfragment;
- and
- (d) correlating the amount of heavy hydrogen label of each subfragment with the amino acid sequence of the protein, thereby localizing the position of the labeled peptide amide group in the protein to within about 1-5 amino acid residues.
A method of determining, at a resolution of about 1-5 amino acid residues, which peptide amide groups in a protein of known amino acid sequence are accessible to solvent, said method comprising the steps of:
- (a) contacting a polypeptide with heavy hydrogen under conditions wherein peptide amide hydrogens in the protein which are freely accessible to solvent exchange with, and become selectively labeled by, heavy hydrogen;
- (b) fragmenting the selectively labeled protein under conditions of slow hydrogen exchange into a plurality of fragments;
- (c) determining, under conditions of slow hydrogen exchange, which of said fragments are labeled with heavy hydrogen;
- (d) progressively degrading each fragment which is labeled with heavy hydrogen under conditions of slow hydrogen exchange into at least one series of subfragments, wherein each subfragment in each of said series is shorter than the preceding subfragment in the series by about 1-5 amino acid residues at one terminus;
- (e) quantifying the amount of heavy hydrogen label on each subfragmcnt;
- and
- (f) correlating the amount of heavy hydrogen label of each subfragment with the amino acid sequence of the fragment from which it has been degraded, thereby localizing the position of the labeled peptide amide in the fragment to within about 1-5 amino acid residues.
A method of determining, at a resolution of about 1-5 amino acid residues, the position of a peptide amide group labeled with a heavy hydrogen in a protein of known amino acid sequence, said method comprising the steps of:
- (a) progressively degrading, under conditions of slow hydrogen exchange, said protein into a mixture of subfragments, wherein said mixture comprises at least one series of subfragments, such that each subfragment in each of said series is shorter than the preceding subfragment in the series by about 1-5 amino acid residues at one terminus and such that the first subfragment in the series is derived from and composed of about 1-5 fewer amino acid residues than said protein and such that the last subfragment in the series is about 1-5 amino acid residues in length;
- and, when said mixture of subfragments comprises an analytically sufficient quantity of each subfragment in each of said series
- (b) quantifying the amount of heavy hydrogen label associated with each subfragment;
- and
- (c) correlating the amount of heavy hydrogen label of each subfragment with the amino acid sequence of the protein, thereby localizing the position of the labeled peptide amide group in the protein to within about 1-5 amino acid residues.
Dependencies are inferred from the claim text.