Methods for microbiological identification by mass spectrometry

Abstract

Methods and systems for identification of microorganisms either after isolation from a culture or directly from a sample. The methods and systems are configured to identify microorganisms based on the characterization of proteins of the microorganisms via high-resolution/mass accuracy single-stage (MS) or multi-stage (MSn) mass spectrometry. Included herein are also discussion of targeted detection and evaluation of virulence factors, antibiotic resistance markers, antibiotic susceptibility markers, typing, or other characteristics using a method applicable to substantially all microorganisms and high-resolution/mass accuracy single-stage (MS) or multi-stage (MSn) mass spectrometry.

What is claimed is: 
     
         1 . A method for identifying and characterizing one or more unknown microbes in a sample, the method comprising:
 (a) providing a fluid extract of the sample, the fluid extract comprising a mixture containing proteins from the one or more unknown microbes;   (b) performing a first, top-down analytical method using mass spectrometry to detect at least one protein from the mixture and optionally identify the at least one protein;   (c) comparing, during a data acquisition period of the first, top-down analytical method, mass spectrometric data for the detected, and optionally identified, at least one protein from step (b) to protein information contained in a reference database of known microbes;   (d) identifying, during the data acquisition period, at least one microbe to the genus species level based on at least one match between the mass spectrometric data and the protein information contained in the reference database; and   (e) performing a second, top-down analytical method using targeted mass spectrometry based on the identified at least one microbe to determine if one or more proteins indicative of antimicrobial resistance, antimicrobial susceptibility and/or virulence, of the identified at least one microbe is present in the sample and, optionally, identifying and/or quantifying the one or more proteins indicative of antimicrobial resistance, antimicrobial susceptibility, and/or virulence,   wherein the first, top-down analytical method includes mass spectrometry analysis of the intact, soluble proteins of the at least one microbe.   
     
     
         2 . The method of  claim 1 , wherein neither the first, top-down analytical method nor the second, top-down analytical method includes chemical or enzymatic digestion of proteins prior to performing mass spectrometry. 
     
     
         3 . The method of  claim 1 , wherein the first, top-down analytical method incorporates or includes direct infusion of the mixture into an electrospray apparatus without chromatographic separation. 
     
     
         4 . The method of  claim 1 , wherein the second, top-down analytical method is selected automatically from a list of analytical methods and based on the identified at least one microbe from step (d). 
     
     
         5 . The method of  claim 1 , wherein the first, top-down analytical method and the second, top-down analytical method are performed using the same mass spectrometer. 
     
     
         6 . The method of  claim 1 , wherein one or both of the first, top-down analytical method and the second, top-down analytical method comprises:
 ionizing intact proteins to form ionized proteins; and   analyzing the ionized proteins using mass spectrometry, wherein analyzing comprises:
 (i) in a first mass spectrometry step, acquiring one or more mass spectra representative of one or more of the ionized proteins; 
 (ii) determining molecular weights for the one or more ionized proteins from the one or more mass spectra; 
 (iii) using the determined molecular weights of the one or more ionized proteins to search a first database containing molecular weights of known microbial proteins, and selecting a subset of candidate microbes from the first database; 
 (iv) in a second mass spectrometry step, selecting one or more precursor ions from the one or more ionized proteins and fragmenting the one or more precursor ions by fragmentation means to produce a plurality of product ions; and 
 (v) using respective mass-to-charge ratios (m/z) of the plurality of product ions to search a second database containing molecular weights of known microbial proteins and at least one of: product ion m/z values; amino acid sequence; or post-translational modification information of said known microbial proteins, wherein the subset of candidate microbes from the first database is used to limit a search of the second database. 
   
     
     
         7 . A method for identifying and characterizing one or more unknown microbes in a sample, the method comprising:
 (a) providing a fluid extract of the sample, the fluid extract comprising intact, soluble proteins from at least one of the one or more unknown microbes;   (b) performing a first, top-down analytical method using mass spectrometry to detect at least one intact, soluble protein and optionally identify the at least one intact, soluble protein, the first, top-down analytical method comprising a data acquisition period in which mass spectrometric data for intact, soluble proteins is obtained;   (c) comparing, during the data acquisition period, mass spectrometric data, information derived from mass spectrometric data, or an identity of the at least one intact, soluble protein to protein information contained in a reference database of known microbes;   (d) identifying, during the data acquisition period, the at least one microbe to the genus species level based on one or more matches between the protein information contained in the reference database and the mass spectrometric data, the information derived from the mass spectrometric data, or the identity of the at least one intact, soluble protein; and   (e) performing a second, top-down analytical method using targeted mass spectrometry based on the identified at least one microbe from step (d) to determine if one or more proteins indicative of antimicrobial resistance, antimicrobial susceptibility and/or virulence of the identified at least one microbe is present in the sample and, optionally, identifying and/or quantifying the one or more proteins indicative of antimicrobial resistance, antimicrobial susceptibility, and/or virulence.   
     
     
         8 . The method of  claim 7 , wherein the first, top-down analytical method incorporates or includes direct infusion of the fluid extract into an electrospray apparatus without chromatographic separation. 
     
     
         9 . The method of  claim 7 , wherein the second, top-down analytical method incorporates chromatographic separation of proteins prior to introducing said proteins into an electrospray apparatus. 
     
     
         10 . The method of  claim 7 , wherein one or both of the first, top-down analytical method and the second, top-down analytical method comprises:
 ionizing intact proteins to form ionized proteins; and   analyzing the ionized proteins using mass spectrometry, wherein analyzing comprises:
 (i) in a first mass spectrometry step, acquiring one or more mass spectra representative of one or more of the ionized proteins; 
 (ii) determining molecular weights for the one or more ionized proteins from the one or more mass spectra; 
 (iii) using the determined molecular weights of the one or more ionized proteins to search a first database containing molecular weights of known microbial proteins, and selecting a subset of candidate microbes from the first database; 
 (iv) in a second mass spectrometry step, selecting one or more precursor ions from the one or more ionized proteins and fragmenting the one or more precursor ions by fragmentation means to produce a plurality of product ions; and 
 (v) using respective mass-to-charge ratios (m/z) of the plurality of product ions to search a second database containing molecular weights of known microbial proteins and at least one of: product ion m/z values; amino acid sequence; or post-translational modification information of said known microbial proteins, wherein the subset of candidate microbes from the first database is used to limit a search of the second database. 
   
     
     
         11 . The method of  claim 7 , wherein neither the first, top-down analytical method nor the second, top-down analytical method includes chemical or enzymatic digestion of proteins prior to performing mass spectrometry. 
     
     
         12 . The method of  claim 7 , wherein the second, top-down analytical method is selected automatically from a list of analytical methods and based on the identified at least one microbe from step (d). 
     
     
         13 . The method of  claim 7 , wherein the first, top-down analytical method and the second, top-down analytical method are performed using the same mass spectrometer. 
     
     
         14 . A method for identifying and characterizing one or more unknown microbes in a sample, the method comprising:
 (a) providing a fluid extract of the sample, the fluid extract comprising intact, soluble proteins from at least one of the one or more unknown microbes;   (b) performing a first, top-down analytical method using mass spectrometry to (i) obtain mass spectrometric data for a first intact, soluble protein from the at least one microbe, (ii) optionally identify the first intact, soluble protein, and (iii) obtain mass spectrometric data for one or more additional intact, soluble proteins from the at least one microbe;   (c) comparing the mass spectrometric data for the first intact, soluble protein, information derived from the mass spectrometric data for the first intact, soluble protein, or an identity of the first intact, soluble protein to protein information contained in a reference database of known microbes;   (d) identifying the at least one microbe to the genus species level based on one or more matches between the protein information contained in the reference database and the mass spectrometric data for the first intact, soluble protein, information derived from the mass spectrometric data for the first intact, soluble protein, or an identity of the first intact, soluble protein; and   (e) performing a second, top-down analytical method using targeted mass spectrometry based on the identified at least one microbe from step (d) to determine if one or more proteins indicative of antimicrobial resistance, antimicrobial susceptibility and/or virulence of the identified at least one microbe is present in the sample and, optionally, identifying and/or quantifying the one or more proteins indicative of antimicrobial resistance, antimicrobial susceptibility, and/or virulence,   wherein steps (c) and (d) are performed while obtaining the mass spectrometric data for the one or more additional intact, soluble proteins.   
     
     
         15 . The method of  claim 14 , wherein the second, top-down analytical method is selected automatically from a list of analytical methods and based on the identified at least one microbe from step (d). 
     
     
         16 . The method of  claim 14 , wherein the first, top-down analytical method and the second, top-down analytical method are performed using the same mass spectrometer. 
     
     
         17 . The method of  claim 14 , wherein one or both of the first, top-down analytical method and the second, top-down analytical method comprises:
 ionizing intact proteins to form ionized proteins; and   analyzing the ionized proteins using mass spectrometry, wherein analyzing comprises:
 (i) in a first mass spectrometry step, acquiring one or more mass spectra representative of one or more of the ionized proteins; 
 (ii) determining molecular weights for the one or more ionized proteins from the one or more mass spectra; 
 (iii) using the determined molecular weights of the one or more ionized proteins to search a first database containing molecular weights of known microbial proteins, and selecting a subset of candidate microbes from the first database; 
 (iv) in a second mass spectrometry step, selecting one or more precursor ions from the one or more ionized proteins and fragmenting the one or more precursor ions by fragmentation means to produce a plurality of product ions; and 
 (v) using respective mass-to-charge ratios (m/z) of the plurality of product ions to search a second database containing molecular weights of known microbial proteins and at least one of: product ion m/z values; amino acid sequence; or post-translational modification information of said known microbial proteins, wherein the subset of candidate microbes from the first database is used to limit a search of the second database. 
   
     
     
         18 . The method of  claim 14 , wherein neither the first, top-down analytical method nor the second, top-down analytical method includes chemical or enzymatic digestion of proteins prior to performing mass spectrometry. 
     
     
         19 . The method of  claim 14 , wherein the first, top-down analytical method incorporates or includes direct infusion of the fluid extract into an electrospray apparatus without chromatographic separation. 
     
     
         20 . The method of  claim 14 , wherein the second, top-down analytical method incorporates chromatographic separation of proteins prior to introducing said proteins into an electrospray apparatus.