Mass Spectrometric Quantitation

Abstract

Provided is a method of assaying for an analyte, which method comprises: a) combining a test sample, which may comprise the analyte, with a calibration sample comprising at least two different aliquots of the analyte, each aliquot having a known quantity of the analyte, wherein the sample and each aliquot are differentially labeled with one or more isobaric mass labels each with a mass spectrometrically distinct mass marker group, such that the test sample and each aliquot of the calibration sample can be distinguished by mass spectrometry; b) determining by mass spectrometry the quantity of the analyte in the test sample and the quantity of the analyte in each aliquot in the calibration sample, and calibrating the quantity of the analyte in the test sample against the known and determined quantities of the analytes in the aliquots in the calibration sample.

1 . A method of assaying for an analyte, which method comprises:
 a) combining a test sample, which may comprise the analyte, with a calibration sample comprising at least two different aliquots of the analyte, each aliquot having a known quantity of the analyte, wherein the sample and each aliquot are differentially labelled with one or more isobaric mass labels each with a mass spectrometrically distinct mass marker group, such that the test sample and each aliquot of the calibration sample can be distinguished by mass spectrometry;   b) determining by mass spectrometry the quantity of the analyte in the test sample and the quantity of analyte in each aliquot in the calibration sample, and calibrating the quantity of the analyte in the test sample against the known and determined quantities of the analytes in the aliquots in the calibration sample.   
   
   
       2 . A method according to  claim 1 , wherein the test sample may comprise a plurality of different analytes and a calibration sample is provided for each different analyte, and wherein step (b) is repeated for each different analyte. 
   
   
       3 . A method according to  claim 2 , wherein the plurality of analytes are peptide fragments of a protein or polypeptide which are produced by chemical or enzymatic processing of the protein or polypeptide prior to step (a). 
   
   
       4 . A method according to  claim 2 , wherein the different aliquots of each calibration sample are selected such that each calibration sample provides a range of quantities of analyte which are different to the range of quantities of analyte in other calibration samples. 
   
   
       5 . A method according  claim 1 , wherein a plurality of test samples are assayed for an analyte. 
   
   
       6 . A method according to  claim 5 , wherein each of the plurality of test samples is assayed for the same analyte. 
   
   
       7 . A method according to  claim 6 , wherein each of the test samples is differentially labelled with one or more of the isobaric mass labels and combined with a single calibration sample in step (a), and the quantity of the analyte in each sample is determined simultaneously in step (b). 
   
   
       8 . A method according to  claim 6 , wherein each test sample is labelled with the same mass label, and steps (a) and (b) are repeated for each different sample. 
   
   
       9 . A method according to  claim 8 , wherein the same calibration sample is used for each test sample to be assayed. 
   
   
       10 . A method according  claim 1 , wherein the method comprises a further step prior to step (a) of differentially labelling each test sample or each aliquot of the calibration sample with one or more isobaric mass labels. 
   
   
       11 . A method according to  claim 10 , which comprises a further step of combining the differentially labelled aliquots to produce a calibration sample prior to step (a). 
   
   
       12 . A method according to  claim 1 , wherein step (b) comprises:
 i) in a mass spectrometer selecting and fragmenting ions of a mass to charge ratio corresponding to the analyte labelled with the mass label, detecting and producing a mass spectrum of fragment ions, and identifying the fragment ions corresponding to the mass marker groups of the mass labels;   ii) determining the quantity of the analyte in each test sample on the basis of the quantity of their mass marker groups in a mass spectrum relative to the quantities of the mass marker groups from the aliquots of the calibration sample in the same mass spectrum.   
   
   
       13 . A method according to  claim 1 , wherein the quantity of an analyte in each aliquot in the calibration sample is a known absolute quantity. 
   
   
       14 . A method according to  claim 13 , wherein the absolute quantity of an analyte in a test sample is determined in step (b). 
   
   
       15 . A method according to  claim 1 , wherein the quantity of analyte in each aliquot in the calibration sample is a known qualitative quantity. 
   
   
       16 . A method according to  claim 15 , wherein the qualitative quantity is an expected range of quantities of analyte in a subject having a particular state. 
   
   
       17 . A method according to  claim 15 , wherein the calibrating step comprises calibrating the quantity of the analyte in the test sample against the known qualitative and determined quantities of the analytes in the aliquots of the calibration sample. 
   
   
       18 . A method according to  claim 17 , wherein the percentage change in the amount of the analyte in the test sample is determined. 
   
   
       19 . A method according to  claim 1 , wherein the quantity of analyte in each different aliquot is selected to reflect the known or suspected variation in the quantity of the analyte in the test sample. 
   
   
       20 . A method according to  claim 19 , wherein aliquots are provided which comprise the analyte in quantities which correspond to the upper and lower limits, and optionally intermediate points within a range of the known or suspected quantities of the analyte found in test samples of healthy or diseased subjects. 
   
   
       21 . A method according to  claim 1 , wherein the different quantities of analyte present in the different aliquots correspond to the known or suspected quantity of analyte present in a test sample which has been incubated for different periods of time. 
   
   
       22 . A method according to  claim 1 , wherein the aliquots are taken from a sample which is a standardised form of the test sample. 
   
   
       23 . A method according to  claim 1 , wherein the test sample and/or the aliquots of the calibration sample are from a plant or an animal. 
   
   
       24 . A method according to  claim 23 , wherein the animal is a human. 
   
   
       25 . A method according to  claim 1 , wherein the calibration sample comprises an analyte in a quantity that indicates the efficacy and/or toxicity of a therapy. 
   
   
       26 . A method according to  claim 1 , wherein the test sample and/or the calibration sample comprises human or animal tissue, blood, plasma, serum, cerebrospinal fluid, synovial fluid, ocular fluid, urine, tears, tear duct exudates, lung aspirates, breast milk, nipple aspirate, semen, lavage fluid, cell extract, tissue culture extract, plant tissue, plant fluid, plant cell culture extract, a bacterial sample, a virus sample, fungus, fermentation broth, a foodstuff or a pharmaceutical composition. 
   
   
       27 . A method according to  claim 1 , wherein the analyte comprises a protein, a polypeptide, a peptide, an amino acid or a nucleic acid, a peptide-nucleic acid, a sugar, starch, a complex carbohydrate, a lipid, a polymer, or fragments thereof. 
   
   
       28 . A method according to  claim 1 , which further includes the step of separating the isobarically labelled analytes electrophoretically or chromatographically after step (a) but before step (b). 
   
   
       29 . A method according to  claim 1 , wherein the calibration sample comprises a further aliquot which comprises the analyte in a quantity which serves as a trigger during an MS scan or during non-scanning MS/MS to initiate an MS/MS scan. 
   
   
       30 . A method according to  claim 29 , wherein the analyte in the further aliquot is labelled with an isobaric mass label. 
   
   
       31 . A method according to  claim 29 , wherein the analyte in the further aliquot is labelled with a mass label which is chemically identical to but isotopically distinct and differing in mass from the isobaric mass labels of the other analytes in the calibration sample 
   
   
       32 . A method according to  claim 1 , wherein the analyte in the sample is a protein, and the analyte in the calibration sample is a recombinant form of the protein in the sample. 
   
   
       33 . A method according to  claim 1 , wherein the mass label comprises the following structure:
   X-L-M   
     wherein X is a mass marker moiety comprising the following group: 
     
       
         
         
             
             
         
       
     
     wherein the cyclic unit is aromatic or aliphatic and comprises from 0-3 double bonds independently between any two adjacent atoms; each Z is independently N, N(R 1 ), C(R 1 ), CO, CO(R 1 ), C(R 1 ) 2 , O or S; X is N, C or C(R 1 ); each R 1  is independently H, a substituted or unsubstituted straight or branched C 1 -C 6  alkyl group, a substituted or unsubstituted aliphatic cyclic group, a substituted or unsubstituted aromatic group or a substituted or unsubstituted heterocyclic group; and y is an integer from 0-10, L is a cleavable linker and M is a mass normalisation moiety. 
   
   
       34 . A method according to  claim 33 , wherein the cleavable linker attaching the mass marker moiety to the mass normalisation moiety is a linker cleavable by collision. 
   
   
       35 . A method according to  claim 34 , wherein the linker is cleavable by CID, ETD, ECD or SID using mass spectrometry. 
   
   
       36 . A method according to  claim 10 , wherein the labelling step comprises a step of reacting the analyte with a reactive mass label, wherein the reactive mass label comprises a mass label and a reactive functionality. 
   
   
       37 . A method according to  claim 36 , wherein the reactive functionality is capable of reacting with any amino group on a polypeptide and comprises a nucleophile or an electrophile. 
   
   
       38 . A method according to  claim 36 , wherein the reactive functionality comprises the following group: 
     
       
         
         
             
             
         
       
     
     wherein each R 2  is independently H, a substituted or unsubstituted straight or branched C 1 -C 6  alkyl group, a substituted or unsubstituted aliphatic cyclic group, a substituted or unsubstituted aromatic group or a substituted or unsubstituted heterocyclic group. 
   
   
       39 . A method according to  claim 33 , wherein the mass label is a mass label from a set of two or more mass labels,
 wherein each mass normalisation moiety ensures that a mass label has a desired aggregate mass, and wherein the set comprises:   mass labels having a mass marker moiety, each mass marker moiety having a mass different from that of all other mass marker moieties in the set, and each label in the set having a common aggregate mass.   and wherein all the mass labels in the set are distinguishable from each other by mass spectroscopy.   
   
   
       40 . A method according to  claim 39 , wherein each mass label in the set has a mass adjuster moiety, selected from:
 (a) an isotopic substituent situated within the mass marker moiety and/or within the mass normalisation moiety, and   (b) substituent atoms or groups attached to the mass marker moiety and/or attached to the mass normalisation moiety.   
   
   
       41 . A method according to  claim 40 , wherein the mass adjuster moiety is selected from a halogen atom substituent, a methyl group substituent, and  2 H,  15 N,  13 C or  18 O isotopic substituents. 
   
   
       42 . A method according to  claim 41 , wherein the mass adjuster moiety is  15 N or  13 C and the set comprises two mass labels having the following structures: 
     
       
         
         
             
             
         
       
     
   
   
       43 . A method according to  claim 41 , wherein the mass adjuster moiety is  15 N and  13 C and the set comprises five mass labels having the following structures: 
     
       
         
         
             
             
         
       
     
   
   
       44 . A method according to  claim 41 , wherein the mass adjuster moiety is  15 N and  13 C and the set comprises six mass labels having the following structures: 
     
       
         
         
             
             
         
       
     
   
   
       45 . A calibration sample for use in a mass spectrometric assay for an analyte which comprises at least two different aliquots of the analyte, wherein each aliquot is differentially labelled with an isobaric mass label as defined in  claim 33 . 
   
   
       46 . A method of assaying for an analyte, which method comprises:
 a) optionally preparing an isobarically labelled reference material containing a reference biomolecule or mixture of reference biomolecules by reacting with a set of mass labels, each mass label with a mass spectrometrically distinct reporter group;   b) labelling a sample in which the quantity of the biomolecule or mixture of biomolecules is to be quantified by reacting with one of the same set of mass labels as used in step a);   c) adding a known amount of the isobarically labelled reference material into the isobarically labelled test sample prepared in step b);   
     d) optionally separating the isobarically labelled biomolecules electrophoretically or chromatographically;
 e) ionising the labelled biomolecules in a mass spectrometer; 
 f) selecting ions of a predetermined mass to charge ratio corresponding to the mass to charge ratio of the preferred ions of the labelled biomolecule in a mass analyser; 
 g) inducing dissociation of these selected ions by collision or electron transfer; 
 h) detecting the collision products to identify collision product ions that are indicative of the mass labels; 
 i) producing a standard curve of ion intensity versus biomolecule amount based on intensity of the collision product ions that are indicative of the mass labels; 
 j) calculating the absolute or relative abundance of the biomolecule or mixture of biomolecules.