High throughput functional proteomics

Abstract

A method is disclosed which provides a high throughput method for assigning plausible functions to unknown sequence entries in a particular database. The method was used herein to identify lectin proteins which can be found in specific tissues of the rice plant.

1 - 9 . (Canceled)  
     
     
         10 . A method of ascribing a function to a protein, said method comprising: 
 (a) providing a composition comprising one or more proteins;    (b) applying said composition to a functional affinity column;    (c) eluting a bound protein from said functional affinity column;    (d) preparing said eluted protein for mass spectrometry;    (e) analyzing at least a portion of said eluted protein by mass spectrometry thereby producing spectral information;    (f) identifying said eluted protein by matching said spectral information with a theoretical mass spectrum of a protein having a known sequence; and    (g) ascribing a function to said identified protein based on the affinity chromatographic behavior of said identified protein.    
     
     
         11 . The method of  claim 10 , wherein preparing said eluted protein for mass spectrometry comprises subjecting said eluted protein to proteolysis and one or more dimensional chromatography.  
     
     
         12 . The method of  claim 11 , wherein said one or more dimensional chromatography is performed using a high performance liquid chromatography column comprising a strong anion exchange resin followed by a reverse phase resin.  
     
     
         13 . The method of  claim 10 , wherein said composition is a protein extract.  
     
     
         14 . The method of  claim 13 , wherein said protein extract is from a tissue or cell.  
     
     
         15 . The method of  claim 14 , wherein said cell is a microbe.  
     
     
         16 . The method of  claim 14 , wherein said cell is a parasite.  
     
     
         17 . The method of  claim 14 , wherein said cell is a cancer cell.  
     
     
         18 . The method of  claim 13 , wherein said protein extract is fractionated prior to application to said functional affinity column.  
     
     
         19 . The method of  claim 10 , wherein said functional affinity column comprises a small molecule.  
     
     
         20 . The method of  claim 19 , wherein said small molecule is a pharmacophore.  
     
     
         21 . (Canceled)  
     
     
         22 . The method of  claim 10 , wherein said functional affinity column comprises a ligand selected from the group consisting of ATP, phosphate, ECM, metal ion, and enzymatic domain.  
     
     
         23 . (Canceled)  
     
     
         24 . The method of  claim 10 , wherein said bound protein is eluted from said functional affinity column in a single step.  
     
     
         25 . The method of  claim 10 , wherein said bound protein is eluted from said functional affinity column using a stepwise or continuous gradient.  
     
     
         26 . The method of  claim 10 , wherein said mass spectrometry is tandem mass spectrometry.  
     
     
         27 . The method of  claim 10 , wherein the sequence of said protein having a known sequence is present in a database.  
     
     
         28 . The method of  claim 27 , wherein the sequence of said protein having a known sequence is derived from a nucleic acid.  
     
     
         29 . The method of  claim 27 , wherein said protein having a known sequence has an unidentified function.  
     
     
         30 - 31 . (Canceled)  
     
     
         32 . A method of ascribing a function to a protein, said method comprising: 
 (a) providing a composition comprising one or more proteins;    (b) applying said composition to a functional affinity column comprising a ligand, wherein said ligand is a peptide or protein domain;    (c) eluting a bound protein from said functional affinity column;    (d) preparing said eluted protein for mass spectrometry;    (e) analyzing at least a portion of said eluted protein by mass spectrometry thereby producing spectral information;    (f) identifying said eluted protein by matching said spectral information with a theoretical mass spectrum of a protein having a known sequence; and    (g) ascribing a function to said identified protein based on the functional affinity chromatographic behavior of said identified protein.    
     
     
         33 . The method of  claim 32 , wherein preparing said eluted protein for mass spectrometry comprises subjecting said eluted protein to proteolysis and one or more dimensional chromatography.  
     
     
         34 . The method of  claim 33 , wherein said one or more dimensional chromatography is performed using a high performance liquid chromatography column comprising a strong anion exchange resin followed by a reverse phase resin.  
     
     
         35 . The method of  claim 32 , wherein said composition is a protein extract.  
     
     
         36 . The method of  claim 35 , wherein said protein extract is from a tissue or cell.  
     
     
         37 . The method of  claim 36 , wherein said cell is a microbe.  
     
     
         38 . The method of  claim 36 , wherein said cell is a parasite.  
     
     
         39 . The method of  claim 36 , wherein said cell is a cancer cell.  
     
     
         40 . The method of  claim 35 , wherein said protein extract is fractionated prior to application to said functional affinity column.  
     
     
         41 . The method of  claim 32 , wherein said ligand is a cell surface peptide.  
     
     
         42 . The method of  claim 32 , wherein said bound protein is eluted from said functional affinity column in a single step.  
     
     
         43 . The method of  claim 32 , wherein said bound protein is eluted from said functional affinity column using a stepwise or continuous gradient.  
     
     
         44 . The method of  claim 32 , wherein said mass spectrometry is tandem mass spectrometry.  
     
     
         45 . The method of  claim 32 , wherein the sequence of said protein having a known sequence is present in a database.  
     
     
         46 . The method of  claim 45 , wherein the sequence of said protein having a known sequence is derived from a nucleic acid.  
     
     
         47 . The method of  claim 45 , wherein said protein having a known sequence has an unidentified function.