Method of mass spectrometry

Abstract

A method of identifying molecules of biological origin is disclosed. The molecules are identified and the basis of the accurately determined mass to charge ratio of the molecules and at least a further physico-chemical property such as elution time or charge state. Further physico-chemical properties may be used. The experimentally determined accurate mass and physico-chemical properties can then be compared with a look-up table of information. The look-up table may generated or physico-chemical properties of data in a conventional database may be calculated. The ability to recognise and preferably identify the same molecules in two different samples may be used to determine whether a particular biological molecules has been expressed differently in an experimental sample relative to a control sample.

1 - 106 . (canceled)  
   
   
       107 . A method of mass spectrometry comprising: 
 providing a first sample comprising a first mixture of molecules of biological origin;    measuring a first physico-chemical property other than mass to charge ratio of first molecules in said first mixture;    mass analysing said first molecules in said first mixture and accurately determining the mass to charge ratio of said first molecules in said first mixture;    providing a second sample comprising a second mixture of molecules of biological origin;    measuring a first physico-chemical property other than mass to charge ratio of first molecules in said second mixture;    mass analysing said first molecules in said second mixture and accurately determining the mass to charge ratio of said first molecules in said second mixture;    determining the intensity of said first molecules in said first mixture and the intensity of said first molecules in said second mixture, said first molecules in said first mixture and said first molecules in said second mixture having been determined to have substantially the same mass to charge ratio and substantially the same first physico-chemical property; and then    identifying said first molecules in said first mixture and/or said first molecules in said second mixture.    
   
   
       108 . A method as claimed in  claim 107 , wherein said first mixture and/or said second mixture comprise a plurality of different biopolymers, proteins, peptides, polypeptides, oligionucleotides, oligionucleosides, amino acids, carbohydrates, sugars, lipids, fatty acids, vitamins, hormones, portions or fragments of DNA, portions or fragments of cDNA, portions or fragments of RNA, portions or fragments of mRNA, portions or fragments of tRNA, polyclonal antibodies, monoclonal antibodies, ribonucleases, enzymes, metabolites, polysaccharides, phosphorolated peptides, phosphorolated proteins, glycopeptides, glycoproteins or steroids.  
   
   
       109 . A method as claimed in  claim 107 , wherein said first mixture and/or said second mixture comprise at least 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 molecules having different identities.  
   
   
       110 . A method as claimed in  claim 107 , wherein said first mixture and/or said second mixture comprise a non-equimolar heterogeneous complex mixture.  
   
   
       111 . A method as claimed in  claim 107 , wherein the mass to charge ratio of said first molecules in said first mixture and/or the mass to charge ratio of said first molecules in said second mixture is determined to within 20 ppm, 19 ppm, 18 ppm, 17 ppm, 16 ppm, 15 ppm, 14 ppm, 13 ppm, 12 ppm, 11 ppm, 10 ppm, 9 ppm, 8 ppm, 7 ppm, 6 ppm, 5 ppm, 4 ppm, 3 ppm, 2 ppm, 1 ppm or <1 ppm.  
   
   
       112 . A method as claimed in  claim 107 , wherein the mass to charge ratio of said first molecules in said first mixture and/or the mass to charge ratio of said first molecules in said second mixture is determined to within 15-20 ppm, 10-15 ppm, 5-10 ppm or 1-5 ppm.  
   
   
       113 . A method as claimed in  claim 107 , wherein the mass to charge ratio of said first molecules in said first mixture and/or the mass to charge ratio of said first molecules in said second mixture is determined to within 0.01 mass units, 0.009 mass units, 0.008 mass units, 0.007 mass units, 0.006 mass units, 0.005 mass units, 0.004 mass units, 0.003 mass units, 0.002 mass units, 0.001 mass units or <0.001 mass units.  
   
   
       114 . A method as claimed in  claim 107 , further comprising at least one of the following steps: 
 (i) comparing the intensity of said first molecules in said first mixture with the intensity of second molecules in said first mixture;    (ii) comparing the intensity of said first molecules in said first mixture with the intensity of said first molecules in said second mixture;    (iii) comparing the intensity of second molecules in said first mixture with the intensity of second molecules in said second mixture;    (iv) comparing the intensity of said first molecules in said second mixture with the intensity of second molecules in said second mixture; and    (v) comparing the ratio of: (a) the intensity of said first molecules in said first mixture to the intensity of said first molecules in said second mixture with (b) the intensity of second molecules in said first mixture to the intensity of second molecules in said second mixture.    
   
   
       115 . A method as claimed in  claim 114 , wherein said second molecules in said first mixture are substantially the same as said second molecules in said second mixture.  
   
   
       116 . A method as claimed in  claim 114 , wherein said second molecules are endogenous or exogenous to said first and second mixtures.  
   
   
       117 . A method as claimed in  claim 107 , wherein said first physico-chemical property is selected from the group consisting of: (i) elution time, hydrophobicity, hydrophilicity, migration time, or chromatographic retention time; (ii) solubility; (iii) molecular volume or size; (iv) net charge, charge state, ionic charge or composite observed charge state; (v) isoelectric point (pI); (vi) dissociation constant (pKa); (vii) antibody affinity; (viii) electrophoretic mobility; (ix) ionisation potential; (x) dipole moment; (xi) hydrogen-bonding capability or hydrogen-bonding capacity; and (xii) ion mobility in gas phase.  
   
   
       118 . A method as claimed in  claim 107 , wherein said method further comprises: 
 measuring a second physico-chemical property other than mass to charge ratio of said first molecules in said first mixture and said first molecules in said second mixture; and    wherein said first molecules in said first mixture and said first molecules in said second mixture are determined to have substantially the same second physico-chemical property.    
   
   
       119 . A method as claimed in any  claim 118 , wherein said second physico-chemical property is selected from the group consisting of: (i) elution time, hydrophobicity, hydrophilicity, migration time, or chromatographic retention time; (ii) solubility; (iii) molecular volume or size; (iv) net charge, charge state, ionic charge or composite observed charge state; (v) isoelectric point (pI); (vi) dissociation constant (pKa); (vii) antibody affinity; (viii) electrophoretic mobility; (ix) ionisation potential; (x) dipole moment; (xi) hydrogen-bonding capability or hydrogen-bonding capacity; and (xii) ion mobility in gas phase.  
   
   
       120 . A method as claimed in  claim 118 , wherein said method further comprises: 
 measuring a third physico-chemical property other than mass to charge ratio of said first molecules in said first mixture and said first molecules in said second mixture; and    wherein said first molecules in said first mixture and said first molecules in said second mixture are determined to have substantially the same third physico-chemical property.    
   
   
       121 . A method as claimed in  claim 120 , wherein said third physico-chemical property is selected from the group consisting of: (i) elution time, hydrophobicity, hydrophilicity, migration time, or chromatographic retention time; (ii) solubility; (iii) molecular volume or size; (iv) net charge, charge state, ionic charge or composite observed charge state; (v) isoelectric point (pI); (vi) dissociation constant (pKa); (vii) antibody affinity; (viii) electrophoretic mobility; (ix) ionisation potential; (x) dipole moment; (xi) hydrogen-bonding capability or hydrogen-bonding capacity; and (xii) ion mobility in gas phase.  
   
   
       122 . A method as claimed in  claim 120 , wherein said method further comprises: 
 measuring a fourth physico-chemical property other than mass to charge ratio of said first molecules in said first mixture and said first molecules in said second mixture; and    wherein said first molecules in said first mixture and said first molecules in said second mixture are determined to have substantially the same fourth physico-chemical property.    
   
   
       123 . A method as claimed in  claim 122 , wherein said fourth physico-chemical property is selected from the group consisting of: (i) elution time, hydrophobicity, hydrophilicity, migration time, or chromatographic retention time; (ii) solubility; (iii) molecular volume or size; (iv) net charge, charge state, ionic charge or composite observed charge state; (v) isoelectric point (pI); (vi) dissociation constant (pKa); (vii) antibody affinity; (viii) electrophoretic mobility; (ix) ionisation potential; (x) dipole moment; (xi) hydrogen-bonding capability or hydrogen-bonding capacity; and (xii) ion mobility in gas phase.  
   
   
       124 . A method as claimed in  claim 122 , wherein said method further comprises: 
 measuring a fifth or yet further physico-chemical property other than mass to charge ratio of said first molecules in said first mixture and said first molecules in said second mixture; and    wherein said first molecules in said first mixture and said first molecules in said second mixture are determined to have substantially the same fifth or yet further physico-chemical property.    
   
   
       125 . A method as claimed in  claim 125 , wherein said fifth or yet further physico-chemical property is selected from the group consisting of: (i) elution time, hydrophobicity, hydrophilicity, migration time, or chromatographic retention time; (ii) solubility; (iii) molecular volume or size; (iv) net charge, charge state, ionic charge or composite observed charge state; (v) isoelectric point (pI); (vi) dissociation constant (pKa); (vii) antibody affinity; (viii) electrophoretic mobility; (ix) ionisation potential; (x) dipole moment; (xi) hydrogen-bonding capability or hydrogen-bonding capacity; and (xii) ion mobility in gas phase.  
   
   
       126 . A method as claimed in  claim 107 , wherein the mass to charge ratio of said first molecules in said first mixture and/or the mass to charge ratio of said first molecules in said second mixture are mass analysed by either: (i) a Fourier Transform (“FT”) mass spectrometer; (ii) a Fourier Transform Ion Cyclotron Resonance (“FTICR”) mass spectrometer; (iii) a Time of Flight (“TOF”) mass spectrometer; (iv) an orthogonal acceleration Time of Flight (“oaTOF”) mass spectrometer; (v) a magnetic sector mass spectrometer; (vi) a quadrupole mass analyser; (vii) an ion trap mass analyser; and (viii) a Fourier Transform orbitrap, an electrostatic Ion Cyclotron Resonance mass spectrometer or an electrostatic Fourier Transform mass spectrometer.  
   
   
       127 . A method as claimed in  claim 107 , wherein either: (i) said first sample is taken from a diseased organism and said second sample is taken from a non-diseased organism; (ii) said first sample is taken from a treated organism and said second sample is taken from a non-treated organism; or (iii) said first sample is taken from a mutant organism and said second sample is taken from a wild type organism.  
   
   
       128 . A method as claimed in  claim 107 , further comprising: 
 providing one or more further samples comprising one or more further mixtures of molecules of biological origin;    measuring a first physico-chemical property other than mass to charge ratio of said first molecules in said one or more further mixtures;    mass analysing said first molecules in said one or more further mixtures and accurately determining the mass to charge ratio of said first molecules in said one or more further mixtures; and    determining the intensity of said first molecules in said one or more further mixtures wherein said first molecules in said first mixture, said first molecules in said second mixture and said first molecules in said one or more further mixtures are determined to have substantially the same mass to charge ratio and substantially the same first physico-chemical property and optionally the same second and/or third and/or fourth and/or fifth or yet further physico-chemical property.    
   
   
       129 . A method as claimed in  claim 128 , further comprising identifying said first molecules in said one or more further mixtures.  
   
   
       130 . A method as claimed in  claim 107 , wherein said first molecules in said first mixture and/or said first molecules in said second mixture are only identified if the intensity of said first molecules in said first mixture differs from the intensity of said first molecules in said second mixture by more than a predetermined amount.  
   
   
       131 . A method as claimed in  claim 107 , wherein said first molecules in said first mixture and/or said first molecules in said second mixture are only identified if the average intensity of a plurality of different molecules in said first mixture differs from the average intensity of a plurality of different molecules in said second mixture by more than a predetermined amount.  
   
   
       132 . A method as claimed in  claim 130 , wherein said predetermined amount is selected from the group consisting of: (i) 1%; (ii) 2%; (iii) 5%; (iv) 10%; (v) 20%; (vi) 50%; (vii) 100%; (viii) 150%; (ix) 200%; (x) 250%; (xi) 300%; (xii) 350%; (xiii) 400%; (xiv) 450%; (xv) 500%; (xvi) 1000%; (xvii) 5000%; and (xviii) 10000%.  
   
   
       133 . A method as claimed in  claim 107 , wherein said step of identifying said first molecules in said first mixture and/or said first molecules in said second mixture comprises comparing said first physico-chemical property and optionally a second and/or third and/or fourth and/or fifth or yet further physico-chemical property and said determined mass to charge ratio of said first molecules in said first mixture and/or said first molecules in said second mixture with an index of molecules, wherein said index comprises: 
 (i) the identity of each indexed molecule;    (ii) an experimentally determined or predicted first physico-chemical property of each indexed molecule;    (iii) an experimentally determined or predicted accurate mass or mass to charge ratio(s) of each indexed molecule; and    (iv) optionally an experimentally determined or predicted second physico-chemical property of each indexed molecule and/or an experimentally determined or predicted third physico-chemical property of each indexed molecule and/or an experimentally determined or predicted fourth physico-chemical property of each indexed molecule and/or an experimentally determined or predicted fifth or yet further physico-chemical property of each indexed molecule.    
   
   
       134 . A method as claimed in  claim 133 , wherein said first molecules in said first mixture and/or said first molecules in said second mixture comprise a peptide and said index of molecules comprises an index of peptides.  
   
   
       135 . A method as claimed in  claim 134 , wherein said index of peptides was generated by determining how one or more proteins might fragment or be digested so as to result in a plurality of peptides.  
   
   
       136 . A method as claimed in  claim 133 , wherein said first molecules in said first mixture and/or said first molecules in said second mixture comprise a peptide and said index of molecules comprises an index of proteins.  
   
   
       137 . A method as claimed in  claim 107 , wherein said step of identifying said first molecules in said first mixture and/or said first molecules in said second mixture comprises calculating said first and/or second and/or third and/or fourth and/or fifth or yet further physico-chemical properties from an index of molecules, said index comprising: 
 (i) the identity of each indexed molecule; and    (ii) an experimentally determined or predicted accurate mass or mass to charge ratio(s) of each indexed molecule.    
   
   
       138 . A method as claimed in  claim 137 , wherein said index comprises: (i) a protein or proteome sequence database; (ii) an Expressed Sequence Tag (EST) database; or (iii) a gene or genome database.  
   
   
       139 . A method as claimed in  claim 107 , wherein said first molecules in said first mixture and/or said first molecules in said second mixture are identified on the basis of: 
 (i) the closeness of fit of the determined mass to charge ratio of said first molecules in said first mixture and/or said first molecules in said second mixture with the mass or mass to charge ratio of an indexed molecule; and/or    (ii) the closeness of fit of said first physico-chemical property of said first molecules in said first mixture and/or said first molecules in said second mixture with the first physico-chemical property of an indexed molecule; and/or    (iii) the closeness of fit of a second physico-chemical property of said first molecules in said first mixture and/or said first molecules in said second mixture with a second physico-chemical property of an indexed molecule; and/or    (iv) the closeness of fit of a third physico-chemical property of said first molecules in said first mixture and/or said first molecules in said second mixture with a third physico-chemical property of an indexed molecule; and/or    (v) the closeness of fit of a fourth physico-chemical property of said first molecules in said first mixture and/or said first molecules in said second mixture with a fourth physico-chemical property of an indexed molecule; and/or    (vi) the closeness of fit of a fifth or yet further physico-chemical property of said first molecules in said first mixture and/or said first molecules in said second mixture with a fifth or yet further physico-chemical property of an indexed molecule.