Methods for quantification and de novo polypeptide sequencing by mass spectrometry

Abstract

The invention provides a method of determining an amino acid sequence of a parent polypeptide. The method consists of: (a) obtaining mass spectra of two or more differentially labeled polypeptide fragments of a parent polypeptide; (b) assigning a mass and a weighting characteristic to two or more paired signals having a difference in mass corresponding to an integer value of said differential label, the weighting characteristic combining properties of each signal within said paired signals; (c) selecting from the mass spectra a paired signal having the assigned mass and a weighting characteristic distinguishable from non-peptide signals, the assigned mass indicating the mass of a polypeptide fragment within the spectra; (d) determining the difference in mass of the polypeptide fragments; (e) assigning the mass differences a satisfying amino acid name, and (f) orienting the assigned amino acid names. Also provided is a method of determining the amino acid sequence of a polypeptide. The method consists of: (a) constructing a graph from mass spectra of two or more differentially labeled polypeptides, the graph comprising a node with mass m, number of labels n, intensity i, and mass differential of labels δ; (b) creating a node corresponding to a paired signal having masses of about m and about m+nδ, and (c) adding a labeled weighted directed edge to the graph between any two nodes corresponding to a mass of an amino acid, the labeled weighted directed edge combining properties of the paired signals.

1 . A method of determining amino acid sequence of a polypeptide, comprising: 
 (a) constructing a graph from mass spectra of two or more differentially labeled polypeptides, said graph comprising a node with mass m, number of labels n, intensity i, and mass differential of labels δ;    (b) creating a node corresponding to a paired signal having masses of about m and about m+nδ, and    (c) adding a labeled weighted directed edge to said graph between any two nodes corresponding to a mass of an amino acid, said labeled weighted directed edge combining properties of said paired signals.    
     
     
         2 . The method of  claim 1 , further comprising: 
 (a) creating a source node with total mass M, total number of labels N and fixed intensity I s ; and    (b) creating a terminus node with mass 0, minimum number of labels no, and fixed intensity I t ;    
     
     
         3 . The method of  claim 2 , further comprising selecting a path from the source node to the terminus node.  
     
     
         4 . The method of  claim 3 , further comprising computing a priority score for each path through the graph.  
     
     
         5 . The method of  claim 1 , wherein said differential label marks an internal amino acid residue.  
     
     
         6 . The method of  claim 1 , wherein said differential label marks a terminal amino acid residue.  
     
     
         7 . The method of  claim 1 , wherein said differential label marks a terminal and an internal amino acid residue.  
     
     
         8 . The method of  claim 1 , wherein said differentially labeled polypeptides further comprise stable isotopic labels.  
     
     
         9 . The method of  claim 1 , wherein said differentially labeled polypeptides further comprise heavy and light labeled isotopes selected from the group consisting of hydrogen, carbon, oxygen, nitrogen, sulfur and selenium.  
     
     
         10 . The method of  claim 1 , wherein said differentially labeled polypeptides further comprise an unlabeled polypeptide and a labeled polypeptide.  
     
     
         11 . The method of  claim 1 , wherein said polypeptide is labeled in vivo or in vitro.  
     
     
         12 . The method of  claim 1 , wherein said mass spectra are obtained from a mass spectrometry database.  
     
     
         13 . The method of  claim 1 , wherein said mass spectra are of low resolution.  
     
     
         14 . The method of  claim 1 , further comprising masses of amino acid post-translational modifications.  
     
     
         15 . The method of  claim 1 , further comprising adding complement node with mass M-m, and a number of labels N-n+n 0 .  
     
     
         16 . The method of  claim 1 , further comprising including multiple amino acid edges between nodes, said multiple amino acid edges characterizing a degenerate amino acid residue in said polypeptide sequence.  
     
     
         17 . The method of  claim 1 , wherein steps a-c are repeated one or more times.  
     
     
         18 . The method of  claim 1 , wherein steps a-c are performed by an automated process.  
     
     
         19 . A method of determining an amino acid sequence of a polypeptide, comprising: 
 (a) differentially labeling two or more polypeptide mixtures, and    (b) determining an amino acid sequence of a polypeptide within said mixture using the method of  claim 1 .    
     
     
         20 . The method of  claim 19 , wherein said differential label marks an internal amino acid residue.  
     
     
         21 . The method of  claim 19 , wherein said differential label marks a terminal amino acid residue.  
     
     
         22 . The method of  claim 19 , wherein said differential label marks a terminal and an internal amino acid residue.  
     
     
         23 . The method of  claim 19 , wherein said differentially labeled polypeptides further comprise stable isotopic labels.  
     
     
         24 . The method of  claim 19 , wherein said differentially labeled polypeptides further comprise heavy and light labeled isotopes selected from the group consisting of hydrogen, carbon, oxygen, nitrogen, sulfur and selenium.  
     
     
         25 . The method of  claim 19 , wherein said differentially labeled polypeptides further comprise an unlabeled polypeptide and a labeled polypeptide.  
     
     
         26 . The method of  claim 19 , wherein said polypeptide is labeled in vivo or in vitro.  
     
     
         27 . The method of  claim 19 , wherein said mass spectra are obtained from a mass spectrometry database.  
     
     
         28 . The method of  claim 19 , wherein said mass spectra are of low resolution.  
     
     
         29 . The method of  claim 19 , further comprising separating components of said mixture.  
     
     
         30 . A method of determining an amino acid sequence of a parent polypeptide, comprising: 
 (a) obtaining mass spectra of two or more differentially labeled polypeptide fragments of a parent polypeptide;    (b) assigning a mass and a weighting characteristic to two or more paired signals having a difference in mass corresponding to an integer value of said differential label, said weighting characteristic combining properties of each signal within said paired signals;    (c) selecting from said mass spectra a paired signal having said assigned mass and a weighting characteristic distinguishable from non-peptide signals, said assigned mass indicating the mass of a polypeptide fragment within said spectra;    (d) determining the difference in mass of said polypeptide fragments;    (e) assigning said mass differences a satisfying amino acid name, and    (f) orienting said assigned amino acid names.    
     
     
         31 . The method of  claim 30 , wherein said differential label marks an internal amino acid residue.  
     
     
         32 . The method of  claim 30 , wherein said differential label marks a terminal amino acid residue.  
     
     
         33 . The method of  claim 30 , wherein said differential label marks a terminal and an internal amino acid residue.  
     
     
         34 . The method of  claim 30 , wherein said differentially labeled polypeptides further comprise stable isotopic labels.  
     
     
         35 . The method of  claim 30 , wherein said differentially labeled polypeptides further comprise heavy and light labeled isotopes selected from the group consisting of hydrogen, carbon, oxygen, nitrogen, sulfur and selenium.  
     
     
         36 . The method of  claim 30 , wherein said differentially labeled polypeptides further comprise an unlabeled polypeptide and a labeled polypeptide.  
     
     
         37 . The method of  claim 30 , wherein said parent polypeptide is labeled in vivo or in vitro.  
     
     
         38 . The method of  claim 30 , wherein said mass spectra are obtained from a mass spectrometry database.  
     
     
         39 . The method of  claim 30 , wherein said mass spectra are of low resolution.  
     
     
         40 . A method of determining an amino acid sequence of a parent polypeptide, comprising: 
 (a) obtaining a mass spectra of two differentially labeled polypeptide fragments of said parent polypeptide, said differential label marking a terminal residue and at least one internal amino acid residue;    (b) identifying a paired signal from said mass spectra corresponding to an internal amino acid residue, said paired amino acid signal having a difference in mass corresponding to said differential label;    (c) identifying a paired signal from said mass spectra corresponding to said terminal residue, said paired amino acid signal having a difference in mass corresponding to said differential label;    (d) determining the difference in mass of polypeptide fragments corresponding to said identified paired signals;    (e) assigning said mass differences a satisfying amino acid name, and    (f) orienting said assigned amino acid names.    
     
     
         41 . The method of  claim 40 , wherein said differential label marks two or more internal amino acid residues.  
     
     
         42 . The method of  claim 40 , wherein said differential label marks two terminal amino acid residues.  
     
     
         43 . The method of  claim 40 , wherein said differential label marks a terminal and two or more internal amino acid residues.  
     
     
         44 . The method of  claim 40 , wherein said differentially labeled polypeptides further comprise a stable isotopic label.  
     
     
         45 . The method of  claim 40 , wherein said differentially labeled polypeptides further comprise heavy and light labeled isotopes selected from the group consisting of hydrogen, carbon, oxygen, nitrogen, sulfur and selenium.  
     
     
         46 . The method of  claim 40 , wherein said differentially labeled polypeptides further comprise an unlabeled polypeptide and a labeled polypeptide.  
     
     
         47 . The method of  claim 40 , wherein said parent polypeptide is labeled in vivo or in vitro.  
     
     
         48 . The method of  claim 40 , wherein said mass spectra are obtained from a mass spectrometry database.  
     
     
         49 . The method of  claim 40 , wherein said mass spectra are of low resolution.  
     
     
         50 . The method of  claim 40 , further comprising identifying a paired signal corresponding to a different internal residue having an integer difference in mass corresponding to said differential label.  
     
     
         51 . The method of  claim 40 , further comprising identifying a paired signal corresponding to two or more internal amino acid residues having the same integer difference in mass.  
     
     
         52 . The method of  claim 40 , wherein said step of orienting said assigned names further comprises assigning a weighted value to said paired amino acid signals.  
     
     
         53 . The method of  claim 40 , wherein said terminal residue comprises the lowest integer difference in mass.  
     
     
         54 . The method of  claim 40  wherein said terminal residue is a carboxyl terminus.  
     
     
         55 . The method of  claim 40 , wherein said terminal residue is an amino terminus.