Sequencing by mass spectrometry

Abstract

This invention relates to non-radioactive markers that facilitate the detection and analysis of nascent proteins translated within cellular or cell-free translation systems. Nascent proteins containing these markers can be rapidly and efficiently detected, isolated and analyzed without the handling and disposal problems associated with radioactive reagents. Preferred markers are dipyrrometheneboron difluoride (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)dyes.

1 . A method, comprising: 
 a) providing a tRNA molecule and a mass marker;    b) aminoacylating said tRNA molecule with said mass marker to create a misaminoacylated tRNA;    c) introducing said misaminoacylated tRNA into a translation system under conditions such that said marker is incorporated into a nascent protein; and    d) detecting said mass marker by mass spectrometry.    
     
     
         2 . The method of  claim 1 , further comprising after step c) proteolyzing said nascent protein.  
     
     
         3 . The method of  claim 1 , wherein detection comprises identifying a spectrometric peak which correspond to mass of said nascent protein plus the mass of the marker.  
     
     
         4 . The method of  claim 1 , wherein the translation system comprises a cellular or cell-free translation system.  
     
     
         5 . The method of  claim 1 , wherein said mass marker is selected from the group consisting of photocleavable markers, coupling agents and combinations thereof.  
     
     
         6 . The method of  claim 1 , wherein said mass marker comprises a cleavable marker attached to an amino acid of said nascent protein such that cleavage results in an unmodified amino acid.  
     
     
         7 . The method of  claim 1 , wherein said mass marker comprises a cleavable marker attached to an amino acid of said nascent protein such that cleavage results in a modified amino acid.  
     
     
         8 . The method of  claim 1 , wherein said mass marker comprises an amino acid-photocleavable biotin conjugate.  
     
     
         9 . The method of  claim 1 , wherein the nascent protein is selected from recombinant gene products, gene fusion products, enzymes, cytokines, carbohydrate and lipid binding proteins, nucleic acid binding proteins, hormones, immunogenic proteins, human proteins, viral proteins, bacterial proteins, parasitic proteins and fragments and combinations thereof.  
     
     
         10 . A method, comprising: 
 a) providing a tRNA molecule and a mass marker;    b) aminoacylating said tRNA molecule with said mass marker to create a misaminoacylated tRNA;    c) introducing said misaminoacylated tRNA into a translation system under conditions such that said marker is incorporated into a nascent protein;    d) proteolyzing said nascent protein; and    e) detecting said mass marker by mass spectrometry.    
     
     
         11 . The method of  claim 10 , wherein said proteolyzing generates one or more polypeptide fragments comprising said mass marker.  
     
     
         12 . The method of  claim 11 , wherein detection comprises identifying a spectrometric peak which correspond to mass of said polypeptide fragment plus the mass of the marker.  
     
     
         13 . The method of  claim 10 , wherein said mass marker is selected from the group consisting of photocleavable markers, coupling agents and combinations thereof.  
     
     
         14 . The method of  claim 10 , wherein said mass marker comprises a cleavable marker attached to an amino acid of said nascent protein such that cleavage results in an unmodified amino acid.  
     
     
         15 . The method of  claim 11 , wherein said mass marker comprises a cleavable marker attached to an amino acid of said nascent protein such that cleavage results in a modified amino acid.  
     
     
         16 . The method of  claim 11 , wherein said mass marker comprises an amino acid-photocleavable biotin conjugate.  
     
     
         17 . The method of  claim 1 , wherein the translation system comprises a cellular or cell-free translation system.  
     
     
         18 . A method, comprising: 
 a) providing a tRNA molecule and a photocleavable mass marker;    b) aminoacylating said tRNA molecule with said photocleavable mass marker to create a misaminoacylated tRNA;    c) introducing said misaminoacylated tRNA into a translation system under conditions such that said photocleavable mass marker is incorporated into a nascent protein; and    d) exposing said photocleavable mass marker to electromagnetic radiation.    
     
     
         19 . The method of  claim 18 , wherein said photocleavable mass marker is attached to an amino acid of said nascent protein such that said exposing of step (d) results in photocleavage so as to generate an unmodified amino acid.  
     
     
         20 . The method of  claim 18 , wherein said photocleavable mass marker is attached to an amino acid of said nascent protein such that said exposing of step (d) results in photocleavage so as to generate a modified amino acid.