Methods for isotopically labeling biomolecules using mammalian cell-free extracts

Abstract

Methods for producing an isotope-labeled mammalian, including a human, biomolecule, such as polypeptides and proteins, in a cell-free protein synthesis system. A biomolecule standard is produced having at least one isotope different in abundance than that of the naturally occurring isotopes in the biomolecule. Methods for quantifying biomolecules standards expressed using mammalian cell-free extracts are disclosed. Methods for producing such standards, kits, systems and reagents, relating to the use of isotope-labeled biomolecule as quantification standards in mass spectrometric and nuclear magnetic resonance analysis.

What is claimed is: 
     
         1 . A method for quantitating a protein analyte in a sample, the method comprising
 preparing a heavy isotope labeled protein,   providing a known quantity of the heavy isotope labeled protein to the sample to create a mixture, where the labeled protein and the protein analyte comprise at least one portion of sequence identity,   subjecting the mixture to digestion conditions for preparation of resultant peptide fragments, where the digestion results in at least a first labeled peptide fragment and a first peptide fragment, both having the same sequence,   subjecting the prepared mixture to mass spectrometry, and   quantitating the amount of the analyte in the sample using the known amount of the first labeled peptide fragment.   
     
     
         2 . The method of  claim 1 , wherein the heavy isotope labeled protein is a standard and provides at least one of absolute and relative quantitation of the analyte. 
     
     
         3 . The method of  claim 1 , wherein the heavy isotope labeled protein is purified prior to providing to the sample. 
     
     
         4 . The method of  claim 3 , wherein the heavy isotope labeled protein is quantitated either before or after purification. 
     
     
         5 . The method of  claim 1 , wherein the heavy isotope labeled protein, which is provided to the sample prior to digestion, provides a means to assess digestion efficiency. 
     
     
         6 . The method of  claim 1 , wherein the heavy isotope labeled protein is serially diluted before providing to the sample. 
     
     
         7 . The method of  claim 6 , wherein the serially diluted heavy isotope labeled protein is used to generate a standard curve, which allows for absolute quantitation of the analyte. 
     
     
         8 . The method of  claim 1 , wherein the heavy isotope labeled protein is produced either by in vitro translation or in vitro transcription/translation using a mammalian cell-free extract. 
     
     
         9 . The method of  claim 8 , wherein the mammalian cell-free extract is prepared from cells cultured in media containing heavy isotope-labeled amino acids. 
     
     
         10 . The method of  claim 1 , wherein the heavy isotope is an isotope different in abundance than the naturally occurring isotope. 
     
     
         11 . The method of  claim 1 , wherein a plurality of heavy isotope labeled proteins is provided to the sample. 
     
     
         12 . A kit comprising
 a heavy isotope labeled protein, and   instructions for using the heavy isotope labeled protein as an internal standard for mass spectrometry.