Methods for Top-Down Multiplexed Mass Spectral Analysis of Mixtures of Proteins or Polypeptides

Abstract

Applications of ion-ion reaction chemistry are disclosed in which proton transfer reactions (PTR) and real-time data analysis methods are used to (1) simplify complex mixture analysis of samples introduced into a mass spectrometer, and (2) improve resolution and sensitivity for the analysis of large proteins in excess of 50 kDa by removing charge and reducing the collisional cross section.

What is claimed is: 
     
         1 . A method of identifying the presence or absence of a microorganism type in a sample, comprising:
 (a) identifying a list of analyte compounds whose simultaneous presence in the sample is diagnostic of the presence of the microorganism type in the sample, said list of analyte compounds comprising protein compounds, polypeptide compounds or both protein and polypeptide compounds;   (b) extracting, from the sample, a liquid solution comprising a mixture of sample-derived proteins and polypeptides;   (c) introducing at least a first portion of the liquid solution into an ionization source of a mass spectrometer;   (d) generating, from the at least first portion of the liquid solution at the ionization source, positively charged ions of the mixture of compounds, the positively charged ions comprising a plurality of ion species;   (e) isolating at least a first subset of the plurality of ion species, each isolated subset of the at least a first isolated subset comprising a respective mass-to-charge (m/z) ratio range;   (f) generating a plurality of first-generation product ion species from each isolated subset of ion species by causing each said isolated subset of ion species to be reacted, for a predetermined time duration, with reagent anions that, upon reaction, extract protons from each of one or more ion species of said isolated subset of ion species that comprises a protonated molecular species of a protein or polypeptide compound;   (g) generating at least one mass spectrum, using a mass analyzer of the mass spectrometer, of either first-generation product ion species or second-generation product ion species generated by further reaction of the first-generation product ion species;   (h) for each respective analyte compound in the list, performing the steps of:
 (h1) conducting a search of the at least one mass spectrum of either the first-generation or the second-generation product ion species for a set of one or more m/z ratios that are diagnostic of the respective analyte compound; and 
 (h2) identifying the presence of the respective analyte compound within the liquid solution if the set of one or more m/z ratios is identified in the mass spectrum; and 
   (i) identifying the presence of the microorganism type within the sample if the presence of each and every analyte compound of the list of analyte compounds is identified within the liquid solution.   
     
     
         2 . A method as recited in  claim 1 , wherein a performing of the steps (h1) and (h2) is performed concurrently with the performing of one or more of the steps (c) through (g). 
     
     
         3 . A method as recited in  claim 1 , wherein the microorganism type is defined as a particular genus of bacteria and the list of analyte compounds includes a sufficient number of analyte compounds that are diagnostic of the particular genus of bacteria to enable identification of the presence or absence of the particular genus of bacteria in the sample. 
     
     
         4 . A method as recited in  claim 1 , wherein the microorganism type is defined as a particular species of bacteria and the list of analyte compounds includes a sufficient number of analyte compounds that are diagnostic of the particular species of bacteria to enable identification of the presence or absence of the particular species of bacteria in the sample. 
     
     
         5 . A method as recited in  claim 1 , wherein the microorganism type is defined as a particular sub-species of bacteria and the list of analyte compounds includes a sufficient number of analyte compounds that are diagnostic of the particular sub-species of bacteria to enable identification of the presence or absence of the particular sub-species of bacteria in the sample. 
     
     
         6 . A method as recited in  claim 1 , wherein the microorganism type is defined as a particular strain of virus and the list of analyte compounds includes a sufficient number of analyte compounds that are diagnostic of the particular viral strain to enable identification of the presence or absence of the particular viral strain in the sample. 
     
     
         7 . A method as recited in  claim 1 , wherein the microorganism type is defined as a particular strain of virus and the list of analyte compounds includes a sufficient number of analyte compounds that are diagnostic of the particular viral strain to enable identification of the presence or absence of the particular viral strain in the sample. 
     
     
         8 . A method for identifying the presence or absence of a protein or polypeptide analyte compound within a sample comprising a mixture of compounds that includes a plurality of protein compounds or a plurality of polypeptide compounds or pluralities of both protein and polypeptide compounds, the method comprising:
 (a) introducing a portion of the liquid sample into an electrospray ionization source of a mass spectrometer;   (b) forming positively charged ions of the mixture of compounds of the portion of the liquid sample by electrospray ionization, the positively charged ions comprising a plurality of first-generation ion species;   (c) isolating a plurality of subsets of the first-generation ion species comprising respective mass-to-charge (m/z) ratio ranges, wherein each m/z ratio range includes an m/z ratio of an ion species comprising a respective protonation state of the analyte compound;   (d) generating a plurality of first-generation product ion species from the isolated plurality of subsets of the first-generation ion species by causing the isolated plurality of subsets of the first-generation ion species to be reacted, for a predetermined time duration, with reagent anions that, upon reaction, extract protons from each ion species that comprises a respective protonation state of the analyte compound;   (e) generating a mass spectrum of the first-generation product ion species; and   (f) identifying either the presence of the analyte compound within the sample if the mass spectrum comprises one or more lines at respective predetermined ink ratios that comprise respective intensities above a predetermined threshold or the absence of the analyte compound within the sample otherwise.   
     
     
         9 . A method as recited in  claim 8 , further comprising repeatedly performing steps (a) through (f) a plurality of times, wherein each repetition of step (a) comprises introducing, into the electrospray ionization source, an eluate from a chromatographic column corresponding to a respective retention time. 
     
     
         10 . A method as recited in  claim 8 , wherein the step (f) further comprises determining, if the mass spectrum comprises one or more lines at respective predetermined ink ratios that comprise respective intensities above a predetermined threshold, a quantity or concentration of the analyte compound within the sample based on the one or more intensities. 
     
     
         11 . A method as recited in  claim 8 , further comprising, after the step (b) of forming positively charged ions and prior to the step (c) of isolating a plurality of subsets of the first-generation ion species, the steps of:
 (b1) isolating a subset of the first-generation ion species comprising a randomly-selected mass-to-charge (m/z) ratio range;   (b2) generating a plurality of product ion species from the isolated subsets of the first-generation ion species by causing the isolated subset of the first-generation ion species to be reacted with reagent anions that, upon reaction, extract protons from each ion species that comprises a respective protonation state of the analyte compound or a respective protonation state of another protein or polypeptide compound;   (b3) generating a mass spectrum of the product ion species; and   (b4) automatically determining the m/z ratio ranges to be used in the subsequent step (c), based on the mass spectrum of the product ions.   
     
     
         12 . A method as recited in  claim 11 , wherein the step (b4) comprises automatically determining, from the mass spectrum, a set of m/z ratios corresponding to multiply-protonated ion species of the other protein or polypeptide compound. 
     
     
         13 . A method of identifying the presence or absence of a microorganism type in a sample, comprising:
 (a) identifying a list of analyte compounds whose simultaneous presence in the sample is diagnostic of the presence of the microorganism type in the sample, said list of analyte compounds comprising protein compounds, polypeptide compounds or both protein and polypeptide compounds;   (b) extracting, from the sample, a liquid solution comprising a mixture of sample-derived proteins and polypeptides;   (c) for each respective analyte compound in the list, performing the steps of:
 (c1) introducing a portion of the liquid solution into an electrospray ionization source of a mass spectrometer; 
 (c2) forming positively charged ions of the mixture of compounds of the portion of the liquid solution by electrospray ionization, the positively charged ions comprising a plurality of ion species; 
 (c3) isolating a first subset of the ion species comprising a first mass-to-charge (m/z) ratio range that includes an m/z ratio of a particular predetermined multiply-protonated molecular species of the respective analyte compound; 
 (c4) generating a plurality of first-generation product ion species from the isolated first subset of ion species by causing the isolated first subset of ion species to be reacted, for a predetermined time duration, with reagent anions that, upon reaction, extract protons from each of one or more ion species that comprises a protonated molecular species of a protein or polypeptide compound; 
 (c5) generating a mass spectrum, using a mass analyzer, of either the first-generation product ion species or of second-generation product ion species generated from the first-generation product ion species; 
 (c6) conducting a search of the mass spectrum of either the first-generation or the second-generation product ion species for a set of one or more m/z ratios that are diagnostic of the respective analyte compound; and 
 (c7) identifying the presence of the respective analyte compound within the liquid solution if the set of one or more m/z ratios is identified in the mass spectrum; and 
   (d) identifying the presence of the microorganism type within the sample if the presence of each and every analyte compound of the list of analyte compounds is identified within the liquid solution.