Methods for producing and analyzing biopolymer ladders
AssigneesThe Rockefeller University, The Scripps Research Institute, Ciphergen Biosystems, Inc.
InventorsBrian T. Chait, Rong Wang, Stephen B. H. Kent, Steven M. Clark
Life of this patent
Estimating the term…
Abstract
Methods of producing biopolymer ladders and their use to obtain structural information about the biopolymer. The ladders are produced by setting up catalytic cleavage and terminating reactions at the end of biopolymer molecules. The terminating reactions terminate cleavage of a percentage of the biopolymer molecules at each round of cleavage.
Claims51
A method for producing a polypeptide ladder comprising contacting an ensemble of polypeptide molecules with
- (i) a terminating reagent that reacts with the polypeptide molecules to add terminating moieties and
- (ii) a cleavage reagent that removes terminal units from an end of the polypeptide molecules;
- wherein the rate of removing terminal units from polypeptide molecules bearing a terminating moiety is less than the rate of removing terminal units from polypeptide molecules that do not bear a terminating moiety, the polypeptide molecules being contacted with the cleavage and terminating reagents under conditions and in amounts so as to create a collection of polypeptide molecules having at least two members in a nested set.
A polypeptide ladder comprising a collection of polypeptide molecules having at least two members in a nested set wherein the members bear the same terminating moiety and wherein the terminating moiety is added catalytically.
A method of obtaining information about the sequence of amino acids in a polypeptide comprising the steps of:
- a) preparing a polypeptide ladder by contacting an ensemble of polypeptides with
- (i) a terminating reagent that reacts with the polypeptide molecules to add terminating moieties and
- (ii) a cleavage reagent that cleaves a terminal peptide bond of polypeptides thereby removing a terminal amino acid;
- wherein the rate of removing terminal units from polypeptide molecules bearing a terminating moiety is less than the rate of removing terminal amino acids from polypeptides that do not bear a terminating moiety, the polypeptides being contacted with the cleavage and terminating reagents under conditions and in amounts so as to create a collection of polypeptides having at least two members in a nested set;
- b) determining the differences in mass between the members;
- and
- c) determining the identity of amino acids whose masses correlate with the determined mass differences, whereby the order of mass differences in the polypeptide ladder indicates the identity of the amino acids and indicates the sequence of amino acids in the polypeptide.
Dependencies are inferred from the claim text.