Single Molecule Proteomics

Abstract

This disclosure comprises devices and methods for determining the identity of individual protein molecules in a complex mixture.

1 . A method for analysing a protein, the method comprising:
 i. unfolding a protein into a polypeptide;   ii. tagging polypeptide with reagents that recognise specific residues on said polypeptide;   iii. rendering the polypeptide such that the location, distance between and/or order of tag sites along the polypeptide can be resolved by a method of choice; and   iv. detecting the tags.   
     
     
         2 . The method of  claim 1 , wherein step iii is performed before step ii. 
     
     
         3 . The method of  claim 1 , wherein step ii is performed before step iii. 
     
     
         4 . A method according to  claim 1  where the unfolding is conducted via a, mechanical, chemical or enzymatic method or a combination thereof. 
     
     
         5 . A method according to  claim 1  where the unfolding is facilitated by a molecular motor comprising ClpX, ClpXP. 
     
     
         6 . A method for analysing a polypeptide, the method comprising:
 i. tagging polypeptide with reagents that recognise specific residues on said polypeptide   ii. rendering the polypeptide such that the location, distance between and/or order of tagging sites along the polypeptide is resolvable by tag detection;   iii. detecting the tags.   
     
     
         7 . The method of  claim 6 , wherein step ii is performed before step i. 
     
     
         8 . The method of  claim 6 , wherein step i is performed before step ii. 
     
     
         9 . The method of  claim 6  where the distance between tagging sites is determined via the time elapsed between detection of tags. 
     
     
         10 . A method according to  claim 1 , wherein the tagging is conducted by chemical or biological means. 
     
     
         11 . A method according to  claim 10 , wherein the tagging is performed using, NHS ester, malemide, nickel-histidine chemistry or other technique. 
     
     
         12 . A method according to  claim 10  where the tagging is performed using antibodies, affybodies, or aptamers. 
     
     
         13 . A method according to  claim 10 , wherein the tagging comprises associating the polypeptide with a detectable label. 
     
     
         14 . A method according to  claim 13 , wherein detectable label comprises a fluorescent/luminescent label, a light scattering label, a contrast label, size label or a nanoparticle label. 
     
     
         15 . A method for analysing a protein, the method comprising:
 i. unfolding a protein into a polypeptide   ii. rendering the polypeptide such that the location, distance between and/or order of sites of choice along the polypeptide is resolvable by detecting features of the polypeptide along its length; and   iii. detecting features of the polypeptide along the polypeptide length.   
     
     
         16 . A method according to  claim 1 , wherein the polypeptide is passed linearly through a nano-constriction/gap or nanopore. 
     
     
         17 . A method according to  claim 1 , wherein the features along the length are detected by perturbation of the interaction of two or more entities comprising FRET, RET, electron tunneling/transfer donor and acceptor or semi-conductor source and drain. 
     
     
         18 . A method according to  claim 1 , wherein the experimentally derived location, distance between and/or order of features or labels along each polypeptide length are compared to an in silico database of location, distance between and/or order of features or labels along known polypeptides. 
     
     
         19 . A method according to  claim 18 , wherein a substantial match to an entry in the in silico database reveals the identity of the protein. 
     
     
         20 . A method according to  claim 15 , wherein the sites along the polypeptide are resolved by nanopore/nanogap detection. 
     
     
         21 - 53 . (canceled)