Quantitative peptide analysis by mass spectrometry

Abstract

A method for determining the concentration ratio in a sample of a target peptide to a reference peptide that is chemically identical with the target peptide, but labeled by isotopes, acquires mass spectra of the target and reference peptides. One of a plurality of families of superimposed bell-shaped curves which is a best fit to ion current peak groups of the target and reference peptides in the mass spectra is determined by varying parameters of the families. In each family, each bell-shaped curve has a predetermined height, the curves have fixed distances from each other and the relative curve heights and curve distances in the families are individually calculated from an elemental composition of the peptides and an isotope abundance distribution of elements composing the peptides, taking into account purity of the isotopes. The concentration ratio is then determined from the parameters of the best fit.

What is claimed is: 
     
         1 . A method for determining the concentration of a target biomolecule in a sample, wherein a reference biomolecule is added in a known amount to a known amount of the sample, the reference biomolecule being chemically identical to the target biomolecule and isotopically labeled, the method comprising:
 (a) extracting the target biomolecule and the reference biomolecule from the sample;   (b) acquiring a mass spectrum of the extracted target and reference biomolecules in a mass spectrometer, the mass spectrum having ion peak distributions specific to the target and reference biomolecules, respectively;   (c) repeating the mass spectrum acquisition if the height of the ion peak distribution of either the target or reference biomolecule is less than a respective first or second predetermined quality parameter, and adding the result to the previously acquired spectrum.   
     
     
         2 . The method according to  claim 1 , wherein the quality parameter is the signal-to-noise ratio. 
     
     
         3 . The method according to  claim 1 , wherein the height of the ion peak distribution is deter-mined by fitting a first family of superimposed bell-shaped curves to the ion peak distribution of the target peptide and a second family of superimposed bell-shaped curves to the ion peak distribution of the reference peptide by varying at least one parameter of the families to find fitted parameter values that minimize differences between the families and the respective ion peak distributions, wherein, in each family, each bell-shaped curve has a predetermined relative height and the curves have fixed distances from each other. 
     
     
         4 . The method according to  claim 3 , wherein varying at least one parameter of the families comprises varying a height parameter that changes the height of all bell-shaped curves of a family. 
     
     
         5 . The method of  claim 3 , wherein varying at least one parameter of the families comprises at least one of: (i) varying a shift parameter to shift each curve family in a spectrum scale direction relative to the ion peak distributions of the target and the reference peptides in order to match peak maxima of ion current peaks and curve peaks; and (ii) shifting the first and second families of bell-shaped curves in a spectrum scale direction relative to the ion peak distributions of the target and the reference peptides, each family having fixed curve height distributions, and the two families being separated in the scale direction by a predetermined distance in order to fit jointly the two curve families. 
     
     
         6 . The method of  claim 3 , wherein varying at least one parameter of the families comprises at least one of:
 varying a width parameter that changes the width of all bell-shaped curves;   varying an additional parameter representing the degree of impurity of isotope atoms used for labeling the reference peptides during the fitting process when the exact isotope abundance distribution is not known, and wherein start values for the fitted parameter values are as near to final values as possible; and   varying an additional parameter for fitting the superimposed bell-shaped curves to the back-ground level of the ion peak distributions of the target peptides and reference peptides, respectively, including prolonging the families of superimposed curves in both directions by straight lines, which are fitted to the background.   
     
     
         7 . The method according to  claim 3 , wherein the relative curve heights and inter curve distances in the families for the target peptide and the reference peptide are individually determined from one or both of (i) previously measured peak profiles of target and reference peptides and (ii) the elemental composition of the peptides and the respective isotope abundance distributions of elements composing the target and reference peptides, taking into account the purity of the isotopes used for labeling. 
     
     
         8 . The method according to  claim 1 , wherein the mass spectrum is acquired by a time-of-flight mass spectrometer. 
     
     
         9 . The method according to  claim 8 , wherein repeating the mass spectrum acquisition comprises forming a sum spectrum of multiple time-of-flight mass spectra. 
     
     
         10 . The method according to  claim 9 , wherein a rate of acquisition of the multiple time-of-flight mass spectra is between 5,000 to 20,000 time-of-flight spectra per second. 
     
     
         11 . The method according to  claim 8 , wherein target biomolecule and the reference biomolecule are ionized by matrix-assisted laser desorption/ionization (MALDI) or electrospray ionization (ESI). 
     
     
         12 . The method according to  claim 1  wherein said repetition of spectrum acquisition is carried out only up to a predetermined maximum number of times. 
     
     
         13 . The method according to  claim 1 , wherein the sample is one of a body fluid, a homogenized tissue sample and a tissue prepared for mass spectrometric MALDI imaging. 
     
     
         14 . The method according to  claim 1 , wherein the target biomolecule is one of a natural peptide, a peptide resulting from a proteolytic process, a drug and a metabolite of a drug. 
     
     
         15 . The method according to  claim 14 , wherein the target biomolecule is a peptide that is extracted from the sample using an antibody or affinity reagent. 
     
     
         16 . The method according to  claim 15 , wherein the antibody or affinity reagent has such specificity that peptides other than the target peptide and reference peptide are also extracted. 
     
     
         17 . The method of  claim 1 , further comprising calibrating the method by performing the meth-od with a plurality of predetermined concentration ratios of target biomolecule and reference biomolecule and deriving a calibration curve relative to predetermined and measured ratios. 
     
     
         18 . The method according to  claim 17 , wherein the steps (a) to (c) are performed each on dilution series of the sample wherein an identical amount of reference substance is added. 
     
     
         19 . The method of  claim 1 , wherein concentrations of a plurality of target biomolecules relative to a plurality of reference biomolecules in a single sample are determined by performing steps (a)-(c) once. 
     
     
         20 . The method of  claim 1 , further comprising fragmenting the target biomolecule and the reference biomolecule and performing steps (a)-(c) on resulting fragments of the target and reference biomolecule ions.