Single Molecule Proteomics

Abstract

This disclosure comprises devices and methods for determining the identity of individual protein molecules in a complex mixture by unfolding the protein into a polypeptide, tagging selected residues on the polypeptide with selected oligonucleotide sequence tags that recognize selected residues on said polypeptide, and then detecting the oligonucleotide sequence tags.

1 . A method for analyzing a protein, the method comprising:
 i. unfolding a protein into a polypeptide;   ii. tagging said polypeptide with reagents that recognize specific residues on said polypeptide;   iii. rendering the polypeptide such that the distance between tag sites along the polypeptide is determined via the time elapsed between detection of tags; 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 . The method according to  claim 1  where the unfolding is conducted via a, mechanical, chemical or enzymatic method or a combination thereof. 
     
     
         5 . The method according to  claim 1  where the unfolding is facilitated by a molecular motor comprising ClpX, ClpXP 
     
     
         6 - 9 . (canceled) 
     
     
         10 . The method according to  claim 1 , wherein the tagging is conducted by chemical or biological means. 
     
     
         11 . The method according to  claim 10 , wherein the tagging is performed using, NHS ester, malemide, or nickel-histidine chemistry. 
     
     
         12 . The method according to  claim 10  where the tagging is performed using antibodies, affybodies, or aptamers. 
     
     
         13 . The method according to  claim 10 , wherein the tagging comprises associating the polypeptide with a detectable label. 
     
     
         14 . The 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 analyzing 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 . The method according to  claim 1 , wherein the polypeptide is passed linearly through a nano-constriction/gap or nanopore. 
     
     
         17 . The 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 - 19 . (canceled) 
     
     
         20 . The method according to  claim 15 , wherein the tag sites along the polypeptide are resolved by nanopore/nanogap detection. 
     
     
         21 - 23 . (canceled) 
     
     
         24 . The method according to  claim 1 , wherein the specific residues are detected by optical microscopy, Raman spectroscopy, SERS/SERRS, lens-free microscopy, X-ray microscopy, electron microscopy, scanning probe microscopy, nanopore/nanogap detection, or near-field optical detection. 
     
     
         25 - 39 . (canceled) 
     
     
         40 . The method according to  claim 1 , wherein the polypeptide is fused with a molecule, not comprising amino acids, prior to rendering the polypeptide such that the distance between tag sites along the polypeptide is determined via the time elapsed between detection of tags. 
     
     
         41 - 42 . (canceled) 
     
     
         43 . A method according to  claim 40 , wherein the molecule, not comprising amino acids, comprises a cholesterol. 
     
     
         44 - 47 . (canceled) 
     
     
         48 . A device for carrying out the method according to  1 , the device comprising a macro interface, a microscale portion configured for containing ligands or for receiving ligands from an external reservoir, and a nanoscale portion configured for detecting or analyzing linear polypeptides. 
     
     
         49 - 53 . (canceled) 
     
     
         54 . The method according to  claim 15 , wherein the polypeptide is passed linearly through a nano-constriction/gap or nanopore. 
     
     
         55 . The method according to  claim 15 , 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.