Expression quantification using mass spectrometry

Abstract

In various aspects, the present teachings provide systems, methods, assays and kits for the absolute quantitation of protein expression. In various aspects, the present teachings provide methods of determining the concentration of one or more proteins of interest in one or more samples of interest. In various aspects, the present teachings provide methods of determining the absolute concentration of one or more isoforms of a protein using standard samples of signature protein fragments and parent-daughter ion transition monitoring (PDITM). In various embodiments, the absolute concentration of multiple isoforms of a biomolecule in a sample, multiple proteins in a biological process, a combination of multiple samples, or combinations thereof, can be determined in a multiplex fashion using the present teachings. In various aspects, provided are methods of assessing the response of a biological system to a chemical agent.

1 . A method for determining the concentration of one or more proteins of interest in two or more samples of interest, comprising the steps of: 
 providing a standard sample comprising a signature peptide for each protein of interest;    selecting a diagnostic daughter ion for each signature peptide;    labeling the one or more proteins of interest in two or more samples of interest with different chemical moieties for each sample, the two or more samples of interest thereby being differentially labeled;    labeling one or more standard samples with a chemical moiety;    combining, to produce a combined sample, at least a portion of the one or more labeled standard samples with at least a portion of two or more differentially labeled samples, the differentially labeled samples being labeled with a different chemical moiety than the one or more labeled standard samples combined therewith;    loading at least a portion of the combined sample on a chromatographic column;    subjecting at least a portion of the eluent from the chromatographic column to multiple reaction monitoring, the transmitted parent ion m/z range of each multiple reaction monitoring scan including a m/z value of one or more of the labeled signature peptides and the transmitted daughter ion m/z range of each multiple reaction monitoring scan including a m/z value one or more of the selected diagnostic daughter ions corresponding to the transmitted labeled signature peptide;    measuring the ion signal of one or more of the selected diagnostic daughter ions using said multiple reaction monitoring; and    determining the concentration of a protein of interest in one or more of the two or more samples of interest based at least on a comparison of the measured ion signal of a selected diagnostic daughter ion corresponding to the protein of interest from a sample of interest to the measured ion signal for the selected diagnostic daughter ion corresponding to the protein of interest from a labeled standard sample.    
     
     
         2 . The method of  claim 1 , wherein the step of selecting a diagnostic daughter ion for each signature peptide comprises selecting the diagnostic daughter ion based on one or more of level of detection (LOD), limit of quantitation (LOQ), linearity of quantitation over a specific dynamic range of concentrations, and combinations thereof.  
     
     
         3 . The method of  claim 1 , wherein the step of labeling proteins of interest in one or more standard samples comprises labeling proteins of interest with an isotopically coded affinity tag, and wherein the step of labeling proteins of interest in different samples comprises labeling proteins of interest with an isotopically coded affinity tag.  
     
     
         4 . The method of  claim 1 , wherein the step of labeling proteins of interest in one or more standard samples comprises labeling proteins of interest with an isobaric tag, and wherein the step of labeling proteins of interest in different samples comprises labeling proteins of interest with an isobaric tag.  
     
     
         5 . The method of  claim 1 , wherein the step of labeling proteins of interest in one or more standard samples comprises labeling proteins of interest with a mass differential tag, and wherein the step of labeling proteins of interest in different samples comprises labeling proteins of interest with a mass differential tag.  
     
     
         6 . The method of  claim 1 , further comprising the step of assessing the response of a biological system to a chemical agent based at least on a comparison of the absolute concentrations of two or more proteins in one or more of the two or more samples to the concentration of two or more corresponding proteins in one or more of the standard samples.  
     
     
         7 . The method of  claim 6 , wherein the chemical agent comprises one or more of a pharmaceutical agent, a pharmaceutical composition, a metabolite, a toxin, or combinations thereof.  
     
     
         8 . The method of  claim 7 , wherein the biological system comprises one or more of a whole organism, a sub-unit of a whole organism, a biological process, a biochemical process, a disease state, a cell line, or models thereof, or combinations thereof.  
     
     
         9 . The method of  claim 1 , further comprising the step of assessing the disease state of a biological system based at least on a comparison of the absolute concentrations of two or more proteins in one or more of the two or more samples to the concentration of two or more corresponding proteins in one or more of the standard samples.  
     
     
         10 . The method of  claim 9 , wherein the biological system comprises one or more of a whole organism, a sub-unit of a whole organism, a biological process, a biochemical process, a disease state, a cell line, or models thereof, or combinations thereof.  
     
     
         11 . The method of  claim 1 , wherein the one or more standard samples comprise a pooled reference sample.  
     
     
         12 . The method of  claim 1 , further comprising the step of subjecting at least a portion of the combined sample to digestion to produce a digested combined sample prior to loading of at least a portion of the combined sample on a chromatographic column, and wherein the portion of the combined sample on a chromatographic column is all or a portion of the digested combined sample.  
     
     
         13 . The method of  claim 12 , wherein the digestion comprises chemical digestion.  
     
     
         14 . The method of  claim 12 , wherein the digestion comprises enzymatic digestion.  
     
     
         15 . The method of  claim 1 , wherein one or more of the one or more of the standard samples are subjected to a digestion prior to being combined with the two or more labeled samples of interest to produce a combined sample.  
     
     
         16 . The method of  claim 15 , wherein the digestion comprises chemical digestion.  
     
     
         17 . The method of  claim 15 , wherein the digestion comprises enzymatic digestion.  
     
     
         18 . The method of  claim 1 , wherein the step of determining the concentration of a protein of interest comprises determining the absolute concentration of the protein of interest.  
     
     
         19 . The method of  claim 1 , the step of determining the concentration of a protein of interest comprises determining the relative concentration of the protein of interest, wherein the labeled standard sample comprises a pooled reference sample.  
     
     
         20 . A method for determining the relative concentration of one or more proteins of interest in two or more sets of samples each set of samples comprising two or more samples of interest, comprising the steps of: 
 providing a standard sample comprising a portion of each sample of interest from the two or more sets of samples;    selecting a diagnostic daughter ion for each signature peptide for each protein of interest;    labeling the one or more proteins of interest in two or more sets of samples with one or more chemical moieties belong to a set of labels;    labeling the standard sample with a chemical moiety from the set of labels different from the one or more chemical moieties used on the two or more sets of samples;    combining, to produce a first combined sample, at least a portion of the labeled standard sample with each labeled sample of interest in a first set of the two or more sets of samples;    loading at least a portion of the first combined sample on a chromatographic column;    subjecting at least a portion of the eluent from the chromatographic column for the first combined sample to multiple reaction monitoring, the transmitted parent ion m/z range of each multiple reaction monitoring scan including a m/z value of one or more of the labeled signature peptides and the transmitted daughter ion m/z range of each multiple reaction monitoring scan including a m/z value one or more of the selected diagnostic daughter ions corresponding to the transmitted labeled signature peptide, and measuring the ion signal of one or more of the selected diagnostic daughter ions using said multiple reaction monitoring;    combining, to produce a second combined sample, at least a portion of the labeled standard sample with each labeled sample of interest in a first set of the two or more sets of samples;    loading at least a portion of the second combined sample on a chromatographic column;    subjecting at least a portion of the eluent from the chromatographic column for the second combined sample to multiple reaction monitoring, the transmitted parent ion m/z range of each multiple reaction monitoring scan including a m/z value of one or more of the labeled signature peptides and the transmitted daughter ion m/z range of each multiple reaction monitoring scan including a m/z value one or more of the selected diagnostic daughter ions corresponding to the transmitted labeled signature peptide, and measuring the ion signal of one or more of the selected diagnostic daughter ions using said multiple reaction monitoring; and    determining the concentration of a protein of interest in one or more of the samples of interest in the first set of samples relative to the concentration of the protein of interest in one or more of the samples of interest in the second set of samples based at least on a comparison of the measured ion signal of a selected diagnostic daughter ion corresponding to the protein of interest to the measured ion signal for the selected diagnostic daughter ion corresponding to the protein of interest from the labeled standard sample.