Protein sequencing method and reagents

Abstract

The invention describes methods and reagents useful for sequencing polypeptide molecules. The method comprises affixing a polypeptide to a substrate and contacting the polypeptide with a plurality of probes. Each probe selectively binds to an N-terminal amino acid or an N-terminal amino acid derivative. Probes bound to the polypeptide molecule are then identified before cleaving the N-terminal amino acid or N-terminal amino acid derivative of the polypeptide. Also provided are methods for the sequencing a plurality of polypeptide molecules in a sample and probes specific for N-terminal amino acids or N-terminal amino acid derivatives.

1 . A method of sequencing a polypeptide comprising:
 a. affixing the polypeptide to a substrate;   b. contacting the polypeptide with a plurality of probes, wherein each probe selectively binds to an N-terminal amino acid or a N-terminal amino acid derivative;   c. detecting the probe bound to the polypeptide molecule, thereby identifying the N-terminal amino acid of the polypeptide;   d. cleaving the N-terminal amino acid or N-terminal amino acid derivative of the polypeptide; and   e. repeating steps (b) to (d) to determine the sequence of at least a portion of the polypeptide.   
     
     
         2 . The method of  claim 1 , wherein the polypeptide is a single polypeptide molecule. 
     
     
         3 . The method of  claim 1 , wherein step b) further comprises derivatizing the N-terminal amino acid of the polypeptide prior to contacting the polypeptide with the plurality of probes. 
     
     
         4 . The method of  claim 1 , wherein step a) comprises affixing the polypeptide to the substrate through a C′-terminal carboxyl group or a side chain functional group of the polypeptide. 
     
     
         5 . The method of  claim 1 , wherein the polypeptide is covalently affixed to the substrate. 
     
     
         6 . (canceled) 
     
     
         7 . The method of  claim 1 , wherein the substrate comprises a plurality of spatially resolved attachment points. 
     
     
         8 . The method of  claim 7 , wherein step a) comprises affixing the polypeptide to a spatially resolved attachment point. 
     
     
         9 . The method of  claim 1 , wherein the plurality of probes comprises:
 a. one or more probes that selectively bind to one of 20 natural proteinogenic amino acids;   b. one or more probes that selectively bind to a post-translationally modified amino acid; or   c. one or more probes that selectively bind to a derivative of a) or b).   
     
     
         10 . The method of  claim 11 , wherein at least one probe comprises an affinity capture reagent and a detectable label. 
     
     
         11 . The method of  claim 10 , wherein the step of detecting the probe bound to the polypeptide comprises detecting the detectable label 
     
     
         12 . The method of  claim 10 , wherein the affinity capture reagent comprises a polypeptide. 
     
     
         13 . (canceled) 
     
     
         14 . (canceled) 
     
     
         15 . The method of  claim 1 , wherein step e) comprises cleaving the N-terminal amino acid or N-terminal amino acid derivative of the polypeptide using Edman degradation. 
     
     
         16 . (canceled) 
     
     
         17 . A method of sequencing a plurality of polypeptide molecules in a sample comprising:
 a. affixing the polypeptide molecules in the sample to a plurality of spatially resolved attachment points on a substrate;   b. contacting the polypeptides with a plurality of probes, wherein each probe selectively binds to an N-terminal amino acid or a N-terminal amino acid derivative;   c. for a plurality of polypeptides molecule that are spatially resolved and affixed to the substrate, identifying the probe bound to each polypeptide;   d. cleaving the N-terminal amino acid or N-terminal amino acid derivative of each of the polypeptides; and   e. repeating steps b) to d) to determine the sequence of at least a portion of one or more of the plurality of polypeptide molecules that are spatially resolved and affixed to the substrate.   
     
     
         18 . The method of  claim 17 , wherein the sample comprises a biological fluid, cell extract or tissue extract. 
     
     
         19 . (canceled) 
     
     
         20 . (canceled) 
     
     
         21 . A probe comprising a variant of a polypeptide wherein the variant binds to an N-terminal amino acid or a N-terminal amino acid derivative with a different selectivity than the polypeptide. 
     
     
         22 . (canceled) 
     
     
         23 . The probe of  claim 21 , wherein the polypeptide is a ClpS polypeptide and the variant of the ClpS polypeptide comprises at least 80% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2. 
     
     
         24 . The probe of  claim 23 , wherein the variant comprises one or more mutations at positions that correspond to residues  12 ,  13 ,  14 ,  16 ,  17 ,  18 ,  21 ,  40 ,  43 ,  44 ,  76  or  77  as set forth in SEQ ID NO: 1. 
     
     
         25 . The probe of  claim 23 , wherein the variant CIpS polypeptide comprises cystein residues at positions that correspond to residues 21 and 40 in SEQ ID NO: 1. 
     
     
         26 . The probe of  claim 21 , wherein the variant comprises a polypeptide with at least 80% sequence identify to the polypeptide sequence as set forth in SEQ ID NO: 2 and wherein the variant selectively binds the N-terminal amino acid tryptophan. 
     
     
         27 . The probe of  claim 21 , further comprising a detectable label.