Methods for Data-Dependent Mass Spectrometry of Mixed Biomolecular Analytes

Abstract

A method for mass spectral analysis of a sample containing a plurality of intact protein molecule species comprises: (a) mass analyzing a plurality of ion species generated from a sample portion; (b) automatically recognizing, for each of at least one intact protein molecule species, a respective subset of m/z ratios corresponding to ion species generated from the each intact protein molecule species; and (c) storing or reporting to a user information relating to each subset of the m/z ratios, wherein step (b) comprises: automatically assigning a tentative charge state to each above-threshold m/z ratio; automatically adjusting the tentative charge to achieve a set of self-consistent assigned charge states; and decomposing the assigned charge states into analyte-specific clusters, each analyte-specific cluster being a one of the subsets of the m/z ratios.

What is claimed is: 
     
         1 . A method for mass spectral analysis of a sample containing a plurality of intact protein molecule species, said method including repeatedly introducing a respective portion of the sample into an ionization source of a mass spectrometer; and using the ionization source to generate, from each sample portion, a population of multi-protonated ions that comprises a plurality of ion species generated from each respective intact protein molecule species of said each portion, each ion species comprising a respective charge state, z, and mass-to-charge (m/z) ratio, the method comprising:
 (a) mass analyzing a plurality of ions generated from a first one of the sample portions, thereby generating a mass spectrum thereof, wherein each ion species generated from a protein is represented in the mass spectrum by a respective m/z ratio and a corresponding respective intensity;   (b) automatically recognizing, for each of two or more intact protein molecule species, a respective subset of the m/z ratios corresponding to a set of ion species generated from said each intact protein molecule species and automatically determining a respective charge state, z, for each m/z ratio of each subset; and   (c) storing or reporting to a user information relating to each subset of the m/z ratios,   wherein the step (b) includes:
 (b1) automatically assigning a tentative charge state to each m/z ratio measured in step (a) that corresponds to an above-threshold intensity; 
 (b2) automatically adjusting the assigned tentative charge states to thereby generate a set of self-consistent assigned charge states; and 
 (b3) decomposing the assigned charge states into analyte-specific clusters of charge states, each analyte-specific cluster being a one of the subsets of the m/z ratios. 
   
     
     
         2 . A method as recited in  claim 1 , wherein:
 the step (b) further includes representing each mass spectrum peak as a respective centroid.   
     
     
         3 . A method as recited in  claim 1 , wherein the repeated introducing of a respective portion of the sample into an ionization source of a mass spectrometer is performed in the absence of prior chromatographic separation of the sample into chromatographic fractions. 
     
     
         4 . A method as recited in  claim 1 , further comprising, after the automatic recognition step (b), the step of:
 determining a molecular weight of an intact protein molecule species from the m/z ratios and determined charge states of a recognized subset of the m/z ratios,   wherein the step (c) further comprises storing or reporting to a user the determined molecular weight.   
     
     
         5 . A method for mass spectral analysis as recited in  claim 1 , wherein the repeated introducing of a respective portion of the sample into an ionization source of a mass spectrometer comprises introducing a continuous flow of eluate discharged from a liquid chromatograph into the ionization source, the method further comprising:
 (d) mass analyzing a plurality of ion species that are generated from a different, second one of the sample portions;   (e) automatically recognizing any additional m/z ratios detected in step (d) that were not observed during execution of step (a);   (f) automatically determining if each additional m/z ratio recognized in step (e) corresponds to an intact protein molecule species for which there exists a previously recognized subset of m/z ratios;   (g) automatically recognizing at least one additional subset of m/z ratios if any additional m/z ratio recognized in step (e) does not correspond to an intact protein molecule species for which there exists a previously recognized subset of m/z ratios;   (h) assigning at least one additional m/z ratio to a one of the additional recognized subsets of m/z ratios; and   (i) storing or reporting to a user information relating each additional subset of the m/z ratios.   
     
     
         6 . A method for mass spectral analysis as recited in  claim 1 , wherein the repeated introducing of a respective portion of the sample into an ionization source of a mass spectrometer comprises repeated ionization of a respective sample portion by laser-assisted desorption and ionization. 
     
     
         7 . A method as recited in  claim 1 , further comprising, after the automatic recognition step (b), the step of:
 determining a respective molecular weight of each of the two or more intact protein molecule species from the m/z ratios and determined charge states of the two or more recognized subsets of the m/z ratios,   wherein the step (c) further comprises storing or reporting to a user the two or more determined molecular weights.   
     
     
         8 . A method as recited in  claim 7 , wherein the step (c) comprises reporting the information relating to each subset of the m/z ratios and information relating to the two or more molecular weights to the user in the form of a displayed graph comprising:
 a horizontal axis scaled in units of m/z ratio;   a vertical axis scaled in units of molecular weight; and   a plurality of points plotted on the graph, each point representing a respective ion species in the mass spectrum for which an m/z value is recognized, wherein a horizontal coordinate of each plotted point represents the recognized m/z value of the respective ion species and a vertical coordinate of each plotted point represents the determined molecular weight of the respective intact protein molecule species.   
     
     
         9 . A method as recited in  claim 7 , wherein the displayed graph further comprises:
 a set of horizontal lines, the vertical coordinate of each horizontal line corresponding to a respective determined molecular weight of a respective intact protein molecule species, wherein each horizontal line connects plotted points representing ion species generated from said respective intact protein molecule species; and   a set of non-horizontal lines, wherein each non-horizontal line connects plotted points representing ion species having a same charge state.   
     
     
         10 . A method as recited in  claim 1  wherein the solution comprising the mixture of the intact protein molecule species is an extract derived from a bacterium, further comprising:
 determining a respective molecular weight of each of two or more of the intact protein molecule species from the m/z ratios and determined charge states of the two or more recognized subsets of the m/z ratios; and 
 determining a bacterium species identification based on the determined two or more molecular weights of the intact protein molecule species. 
 
     
     
         11 . A method as recited in  claim 10 , wherein the repeated introducing of a respective portion of the sample to an ionization source comprises introducing a continuous flow of the bacterium extract to the ionization source during direct infusion of the sample into the mass spectrometer. 
     
     
         12 . A method as recited in  claim 11 , wherein at least one of the sample portions includes four or more unique intact protein molecule species. 
     
     
         13 . A method as recited in  claim 10 , wherein the step (b) is performed in not more than one second. 
     
     
         14 . A method as recited in  claim 10 , wherein the repeated introducing of a respective portion of the sample to an ionization source comprises:
 introducing the bacterium extract into a liquid chromatographic column;   performing a partial chromatographic separation of the intact protein molecule species of the bacterium extract using the liquid chromatographic column, wherein the chromatographic column is operated such that all of the intact protein molecule species elute from the column during a time span of not greater than five minutes; and   introducing a continuous flow of eluate discharged from the liquid chromatographic column to the ionization source, the continuous flow including all of the sample portions.   
     
     
         15 . A method as recited in  claim 14 , wherein at least one of the sample portions includes four or more unique intact protein molecule species. 
     
     
         16 . A method for mass spectral analysis of a sample containing a plurality of intact protein molecule species, said method including repeatedly introducing a respective portion of the sample into an ionization source of a mass spectrometer; and using the ionization source to generate, from each sample portion, a population of multi-protonated ions that comprises a plurality of ion species generated from each respective intact protein molecule species of said each portion, each ion species comprising a respective charge state, z, and mass-to-charge (m/z) ratio, the method comprising:
 (a) mass analyzing a plurality of ions generated from a first one of the sample portions, thereby generating a mass spectrum thereof, wherein each ion species generated from a protein is represented in the mass spectrum by a respective m/z ratio and a corresponding respective intensity;   (b) automatically recognizing, for each of two or more intact protein molecule species, a respective subset of the m/z ratios corresponding to a set of ion species generated from said each intact protein molecule species and automatically determining a respective charge state, z, for each m/z ratio of each subset;   (c) determining a respective molecular weight of each of the two or more intact protein molecule species from the m/z ratios and determined charge states of the two or more recognized subsets of the m/z ratios; and   (d) reporting information relating to each subset of the m/z ratios and information relating to the two or more molecular weights to a user in the form of a displayed graph comprising:
 a horizontal axis scaled in units of m/z ratio; 
 a vertical axis scaled in units of molecular weight; and 
 a plurality of points plotted on the graph, each point representing a respective ion species in the mass spectrum for which an m/z value is recognized, wherein a horizontal coordinate of each plotted point represents the recognized m/z value of the respective ion species and a vertical coordinate of each plotted point represents the determined molecular weight of the respective intact protein molecule species. 
   
     
     
         17 . A method as recited in  claim 16 , wherein the displayed graph further comprises:
 a set of horizontal lines, the vertical coordinate of each horizontal line corresponding to a respective determined molecular weight of a respective intact protein molecule species, wherein each horizontal line connects plotted points representing ion species generated from said respective intact protein molecule species; and   a set of non-horizontal lines, wherein each non-horizontal line connects plotted points representing ion species having a same charge state.   
     
     
         18 . A method as recited in  claim 16 , wherein the solution comprising the mixture of the intact protein molecule species is an extract derived from a bacterium, further comprising:
 determining a bacterium species identification based on the determined two or more molecular weights of the intact protein molecule species.