METHODS FOR IDENTIFYING MODIFICATIONS OF A POLYPEPTIDE
Systems and methods for obtaining qualitative or quantitative measurements of proteoforms of polypeptides are described. The described methods include measurements of affinity reagent binding on single-molecule polypeptide arrays to distinguish between polypeptide isoforms. The described methods may provide high resolution quantitative comparisons of proteoforms with very low copy numbers.
1 . A method for identifying modifications of a first type of polypeptide in a sample, comprising:
providing the first type of polypeptides on an array, wherein individual polypeptide molecules of the first type of polypeptides are located at an individually observable address on the array;
sequentially contacting the array with a plurality of different types of affinity reagents, wherein each of the plurality of different types of affinity reagents has binding specificity for a different modification to the first type of polypeptide; and
characterizing modifications on the individual molecules of the polypeptides based upon the types of different affinity reagents that bind or do not bind to the individual polypeptides on the array.
2 . The method of claim 1 , comprising characterizing patterns of modifications in each of the plurality of individual polypeptides on the array.
3 . The method of claim 2 , comprising quantifying different patterns of modifications to the polypeptides in the sample from quantification of the different patterns of modifications of polypeptides on the array.
4 . The method of claim 1 , wherein the modifications comprise post translational modifications.
5 . The method of claim 4 , wherein the modifications comprise a modification at one or more of a plurality of amino acid residues in the polypeptides, wherein such modifications are independently selected from myristoylation, palmitoylation, isoprenylation, prenylation, farnesylation, geranylgeranylation, lipoylation, flavin moiety attachment, Heme C attachment, phosphopantetheinylation, retinylidene Schiff base formation, dipthamide formation, ethanolamine phosphoglycerol attachment, hypusine, beta-Lysine addition, acylation, acetylation, deacetylation, formylation, alkylation, methylation, C-terminal amidation, arginylation, polyglutamylation, polyglyclyation, butyrylation, gamma-carboxylation, glycosylation, glycation, polysialylation, malonylation, hydroxylation, iodination, nucleotide addition, phosphoate ester formation, phosphoramidate formation, phosphorylation, adenylylation, uridylylation, propionylation, pyrolglutamate formation, S-glutathionylation, S-nitrosylation, S-sulfenylation, S-sulfinylation, S-sulfonylation, succinylation, sulfation, glycation, carbamylation, carbonylation, isopeptide bond formation, biotinylation, carbamylation, oxidation, reduction, pegylation, ISGylation, SUMOylation, ubiquitination, neddylation, pupylation, citrullination, deamidation, elminylation, disulfide bridge formation, proteolytic cleavage, isoaspartate formation, and racemization.
6 . The method of claim 2 , wherein the modifications comprise phosphorylation at one or more amino acid residues in the polypeptides.
7 . The method of claim 6 , wherein the modifications comprise phosphorylation at one or more of a plurality of amino acid residues in the polypeptide, and wherein each of a plurality of the affinity reagents has a binding specificity for a polypeptide that comprises a phosphorylation at a different amino acid residue or set of amino acid residues in the polypeptide.
8 . The method of claim 7 , further comprising characterizing one or more patterns of phosphorylated amino acid residues in the individual polypeptides on the array.
9 . The method of claim 8 , further comprising determining a quantity of different patterns of phosphorylated amino acid residues in the sample from the quantity of different patterns of phosphorylated amino acid residues in the polypeptides on the array.
10 . The method of claim 1 , wherein the modification comprises a splice variation to the type of polypeptide, and each of a plurality of the affinity reagents have a binding specificity to different individual polypeptides having different splice variations.
11 . The method of claim 1 , comprising, following identification of one or more modifications on the polypeptides on the array, treating the polypeptides on the array to remove one or more of the modifications, and repeating the contacting and identify steps to identify polypeptides on the array having the modifications removed.
12 . The method of claim 11 , wherein one or more of the modifications comprise a phosphorylated amino acid residue in the polypeptides, and the treating step comprises contacting the polypeptides with a phosphatase enzyme.
13 . The method of claim 1 , wherein the contacting step comprises sequentially contacting the polypeptides on the array with at least three different affinity reagents.
14 . The method of claim 1 , wherein the contacting step comprises sequentially contacting the polypeptides on the array with at least four different affinity reagents.
15 . The method of claim 1 , wherein the contacting step comprises sequentially contacting the polypeptides on the array with at least six different affinity reagents.
16 . The method of claim 1 , wherein the contacting step comprises sequentially contacting the polypeptides on the array with at least eight different affinity reagents.
17 . The method of claim 1 , wherein at least two different affinity reagents are contacted with the array simultaneously.
18 . The method of claim 1 , further comprising the step of identifying individual locations on the array occupied by the first type of polypeptides.
19 . The method of claim 18 , wherein the step of identifying individual locations on the array occupied by the first type of polypeptides comprises sequentially contacting the polypeptides on the array with a plurality of different affinity reagents, each different affinity reagent having affinity for a different epitope of amino acids, and identifying the polypeptides based upon a pattern of binding of the different affinity reagents to the epitopes in the polypeptides on the array.
20 . A method of characterizing proteoforms of a first type of polypeptide in a sample, comprising:
providing the first type of polypeptides immobilized on an array, wherein individual molecules of the first type of polypeptides are immobilized in individually addressable locations on the array;
separately contacting the polypeptides on the array with each of a plurality of different affinity reagents, each of the plurality of different affinity reagents having binding affinity for a different modification to the first type of polypeptides;
detecting binding of each of the different affinity reagents to individual polypeptide molecules on the array; and
characterizing the proteoforms of the first type of polypeptides in the sample based upon the individual polypeptides on the array that are bound by each of the plurality of different affinity reagents.