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 composition comprising at least a first set of a plurality of peptide isotopologues, where each peptide isotopologue of the first set is present at a different concentration, and has the same amino acid sequence. 
     
     
         22 . The composition of  claim 21  wherein each peptide isotopologue of the first set has the same unit mass. 
     
     
         23 . The composition of  claim 21  further comprising at least one additional set of a plurality of peptide isotopologues, where each additional set comprises a unique peptide sequence where each peptide isotopologue of each additional set is present at a different concentration, and has the same amino acid sequence. 
     
     
         24 . The composition of  claim 23  wherein each peptide isotopologue of the at least one additional set has the same unit mass. 
     
     
         25 . A method for mass spectrometry (MS) calibration, the method comprising
 generating a standard curve for MS analysis based on mass defects using the at least a first set of a plurality of peptide isotopologues of  claim 21 , where each peptide isotopologue of the first set is present at a different concentration, has the same amino acid sequence, and has a same unit mass,   separating the mixed isotopologues with low resolution MS or with high resolution MS, and   using results of the separation to verify the MS calibration.   
     
     
         26 . A method for mass spectrometry (MS) calibration, the method comprising
 generating a dilution curve in a single injection into a liquid chromatograph/mass spectrometer by injecting into a first liquid chromatography (LC) column coupled to a mass spectroscopy detection system a single peptide composition, the single peptide composition comprising a composition comprising at least a first set of a plurality of peptide isotopologues, where each peptide isotopoiogue of the first set has the same amino acid sequence and same unit mass; and each peptide isotopoiogue of the first set is present at a different concentration,   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 dilution curve from the analysis, and   using results of the at least first dilution curve to assess sensitivity of the MS equipment.