Absolute Quantitation of Proteins and Protein Modifications by Mass Spectrometry with Multiplexed Internal Standards

Abstract

A method for absolute protein or peptide quantitation by mass spectroscopy. A sample containing a protein or peptide of interest is prepared for mass spectroscopy analysis. The sample is subjected to mass spectroscopy analysis at low resolution whereby a single additive mass spectroscopy peak is obtained, then is subjected to high resolution mass spectroscopy analysis whereby a plurality of mass spectroscopy peaks are obtained. The intensity of each of the plurality of mass spectroscopy peaks is quantitated either by comparison to an internal standard set, or by using a standard curve generated for each isotopologue set. Quantitation using a standard curve enhances quantitation across a dynamic range of analyte.

What is claimed is: 
     
         1 - 20 . (canceled) 
     
     
         21 . A multiplexed internal standard reference set composition for peptide quantification comprising
 at least one first premixed set of multiplexed internal standard reference peptides comprising a plurality of isolated peptides labeled with heavy isotopes, wherein each peptide of the multiplexed internal standard reference set has the same amino acid sequence and same nominal mass, wherein the nominal mass of each peptide is increased by at least 4 Daltons from the naturally occurring peptide, and the peptides within the set differ by milliDalton mass defects created by incorporating heavy isotopes on different atoms within the amino acid sequence, wherein the heavy isotopes are chosen from  13 C—,  15 N—,  18 O—,  34 S— or  2 H—; and   at least one second premixed set of multiplexed internal standard reference peptides comprising a plurality of isolated peptides labeled with heavy isotopes having a different amino acid sequence from the first set, wherein each peptide of the multiplexed internal standard reference set has the same amino acid sequence and same nominal mass, wherein the nominal mass of each peptide is increased by at least 4 Daltons from the naturally occurring peptide, and the peptides within the set differ by milliDalton mass defects created by incorporating heavy isotopes on different atoms within the amino acid sequence, wherein the heavy isotopes are chosen from  13 C—,  15 N—,  18 O—,  34 S— or  2 H—.   
     
     
         22 . The multiplexed internal standard reference set composition according to  claim 21 , wherein each heavy isotope labeled peptide of the at least one first premixed set of multiplexed internal standard reference peptides is present at a different concentration. 
     
     
         23 . The multiplexed internal standard reference set composition according to  claim 22 , wherein the different concentrations are fixed ratios. 
     
     
         24 . The multiplexed internal standard reference set composition according to  claim 21 , wherein each heavy isotope labeled peptide of the at least one second premixed set of multiplexed internal standard reference peptides is present at a different concentration. 
     
     
         25 . The multiplexed internal standard reference set composition according to  claim 24 , wherein the different concentrations are fixed ratios. 
     
     
         26 . The multiplexed internal standard reference set composition for peptide quantification according to  claim 21 , further comprising a native sample containing the naturally occurring peptide of the first premixed set. 
     
     
         27 . The multiplexed internal standard reference set composition for peptide quantification according to  claim 21 , further comprising a native sample containing the naturally occurring peptide of the second set. 
     
     
         28 . A method for mass spectrometry (MS) calibration, the method comprising
 injecting into a first liquid chromatography (LC) column coupled to a mass spectroscopy detection system a single peptide composition, a multiplexed internal standard reference set comprising at least one first set of a plurality of isolated peptides labeled with heavy isotopes, wherein each heavy isotope labeled peptide of the multiplexed internal standard reference set has the same amino acid sequence and same nominal mass that is increased by at least 4 Daltons from the naturally occurring peptide, and further wherein the peptides within the multiplexed internal standard reference set differs by milliDalton mass defects created by incorporating heavy isotopes on different atoms within at least one amino acid molecule, wherein the heavy isotopes are chosen from  13 C—,  15 N—,  18 O—,  34 S— or  2 H—;   analyzing the multiplexed internal standard mix and corresponding native peptide composition by mass spectroscopy;   generating from the single peptide composition injection into the LC column at least a first calibration curve from the analysis; and   calibrating the MS equipment based on the results of the at least first dilution curve for quantitation of the native peptide.   
     
     
         29 . The method according to  claim 28 , wherein each heavy isotope labeled peptide of the premixed multiplexed internal standard reference set is present at a different concentration. 
     
     
         30 . The method according to  claim 28 , wherein the calibration curve is a standard curve. 
     
     
         31 . The method according to  claim 28 , wherein the calibration curve is a dilution curve. 
     
     
         32 . A method for mass spectrometry (MS) calibration, the method comprising
 generating a calibration curve for MS analysis based on milliDalton mass defects using a multiplexed internal standard reference set composition according to  claim 21 ;   separating the mixed heavy isotope labeled peptides with high resolution MS; and   calibrating the MS equipment based on the results of the separation.   
     
     
         33 . A method for mass spectrometry (MS) calibration, the method comprising
 injecting into a first liquid chromatography (LC) column coupled to a mass spectroscopy detection system a multiplexed internal standard reference set composition according to  claim 21 ;
 analyzing each peptide in the co-eluted peptide composition by mass spectroscopy; 
 generating from the single peptide composition injection into the LC column at least a first calibration curve from the analysis; and 
 calibrating the MS equipment based on the results of the at least first dilution curve for quantitation of the native peptide.