Labeling of proteomic samples during proteolysis for quantitation and sample multiplexing

Abstract

This invention related to methods useful in the labeling of multiple polypeptide samples and subsequent analysis of these samples by mass spectrometry, particularly in the high throughput proteomic setting.

1 - 15 . (canceled)  
     
     
         16 . A method for producing a peptide sample pool for analysis by mass spectrometry, the method comprising: 
 (i) forming a first peptide digest by hydrolyzing a first peptide sample in the presence of water containing a first volumetric ratio of two members of an isotope;    (ii) forming a second peptide digest by hydrolyzing a second peptide sample in the presence of water containing a second volumetric ratio of the said two members of an isotope, wherein said second volumetric ratio is different from said first volumetric ratio; and    (iii) pooling the first peptide digest and the second peptide digest to form a peptide sample.    
     
     
         17 . The method according to  claim 16 , wherein each peptide digest is enzymatically hydrolyzed.  
     
     
         18 . The method according to  claim 17 , wherein the enzyme used for hydrolysis is trypsin.  
     
     
         19 . The method according to  claim 16 , wherein each peptide digest is carried out using chemical means other than enzymatic digestion.  
     
     
         20 . The method according to  claim 16 , wherein the isotope used is selected from either  16 O: 18 O pair or  2 H: 1 H pair.  
     
     
         21 . The method according to  claim 20 , wherein the two members of an isotope is  16 O and  18 O.  
     
     
         22 . The method according to  claim 16 , wherein the volumetric ratio of  16 O: 18 O differs by at least about 5%, preferably 15%, more preferably 30%, and most preferably 90% in terms of the amount of  18 O therein.  
     
     
         23 . A method useful to identify the source of peptides subjected as a peptide sample pool to analysis by mass spectrometry, the method comprising: 
 (i) obtaining a peptide sample pool comprising peptide digests formed by pooling protein digests from at least two different source proteins, wherein each source protein has been hydrolyzed in the presence of water containing an isotope ratio that is different for each protein sample;    (ii) subjecting the peptide sample to analysis by mass spectrometry to generate mass spectra comprising at least one signal doublet for each peptide in the sample, the signal doublet comprising a first signal and a second signal shifted a known units from the first signal, wherein said known units is the difference in molecular weight between the two members of said isotope;    (iii) determining a signal ratio for a given peptide by relating the difference in signal intensity or area between the first signal and the second signal;    (iv) correlating the signal ratio for the given peptide with the isotope ratio used to form the given peptide, thereby identifying the protein source of the given peptide.    
     
     
         24 . The method according to  claim 23 , wherein the peptide sample pool for analysis is generated using the method of  claim 16 .  
     
     
         25 . A peptide sample pool adapted to reveal the protein source of each peptide in the pool when the pool is analyzed by mass spectrometry, the pool comprising peptide digests formed by pooling protein digests from at least two different source proteins, wherein each source protein has been hydrolyzed in the presence of water containing an isotope ratio that is different for each protein sample;  
     
     
         26 . The peptide sample pool of  claim 25 , wherein the peptide sample pool is generated using the methods of  claim 16;   
     
     
         27 . A method useful for tracking in a peptide sample pool the source of every interested polypeptide, comprising: 
 (i) generating a peptide sample pool for analysis by mass spectrometry using the method of  claim 16;     (ii) identifying the source of every interested polypeptide using the method of  claim 23 .    
     
     
         28 . A sofeware program for high throughput automated analysis of mass spectrometry data of peptide sample, comprising: 
 (i) identifying the peptides of interest in the sample with high probability based on their mass data;    (ii) generating a theoretical natural isotope abundance distribution based on said identification of (i);    (iii) subtracting the relative isotopic contribution by each of the labeled states and comapring them in a relative sense to generate the  16 O/ 18 O ration of interest.    
     
     
         29 - 41 . (canceled)  
     
     
         42 . A method for producing a peptide sample pool for analysis by mass spectrometry, the method comprising: 
 (i) forming a first peptide digest by hydrolyzing a first peptide sample in the presence of water containing a first of two members of an isotope;    (ii) forming a second peptide digest by hydrolyzing said first peptide sample in the presence of water containing a second of the said two members of an isotope;    (iii) combining the first peptide digest and the second peptide digest in a predetermined ration to form a first peptide sample;    (iv) repeating (i) to (iii) for a second peptide sample, using a different said predetermined ratio to form a second peptide sample; and,    (v) pooling all peptide samples to form a peptide sample pool.    
     
     
         43 . The method according to  claim 42 , wherein each peptide digest is enzymatically hydrolyzed.  
     
     
         44 . The method according to  claim 43 , wherein the enzyme used for hydrolysis is trypsin.  
     
     
         45 . The method according to  claim 42 , wherein each peptide digest is carried out using chemical means other than enzymatic digestion.  
     
     
         46 . The method according to  claim 42 , wherein the isotope used is selected from either  16 O: 18 O pair or  2 H: 1 H pair.  
     
     
         47 . The method according to  claim 46 , wherein the two members of an isotope is  16 O and  18 O.  
     
     
         48 . The method according to  claim 42 , wherein the volumetric ratio of  16 O: 18 O differs by at least about 5%, preferably 15%, more preferably 30%, and most preferably 90% in terms of the amount of  18 O therein.  
     
     
         49 . A method useful to identify the source of peptides subjected as a peptide sample pool to analysis by mass spectrometry, the method comprising: 
 (i) obtaining a peptide sample pool comprising peptide digests formed by pooling protein digests from at least two different source peptides, wherein each source peptide has been labeled in the presence of water containing an isotope ratio that is different for each protein sample;    (ii) subjecting the peptide sample to analysis by mass spectrometry to generate mass spectra comprising at least one signal doublet for each peptide in the sample, the signal doublet comprising a first signal and a second signal shifted a known units from the first signal, wherein said known units is the difference in molecular weight between the two members of said isotope;    (iii) determining a signal ratio for a given peptide by relating the difference in signal intensity or area between the first signal and the second signal;    (iv) correlating the signal ratio for the given peptide with the isotope ratio used to form the given peptide, thereby identifying the protein source of the given peptide.    
     
     
         50 . The method according to  claim 49 , wherein the peptide sample pool for analysis is generated using the method of  claim 42 .  
     
     
         51 . A peptide sample pool adapted to reveal the protein source of each peptide in the pool when the pool is analyzed by mass spectrometry, the pool comprising peptide digests formed by pooling protein digests from at least two different source proteins, wherein each source protein has been hydrolyzed in the presence of water containing an isotope ratio that is different for each protein sample;  
     
     
         52 . The peptide sample pool of  claim 51 , wherein the peptide sample pool is generated using the methods of  claim 42;   
     
     
         53 . A method useful for tracking in a peptide sample pool the source of every interested polypeptide, comprising: 
 (i) generating a peptide sample pool for analysis by mass spectrometry using the method of  claim 42;     (ii) identifying the source of every interested polypeptide using the method of  claim 49 .    
     
     
         54 - 61 . (canceled)