Methods for identifying bacteria

Patent metadata

Field	Value
Publication number	US-9273339-B2
Application number	US-201213342694-A
Country	US
Kind code	B2
Filing date	Jan 3, 2012
Priority date	Jan 3, 2011
Publication date	Mar 1, 2016
Grant date	Mar 1, 2016

Abstract

The present invention provides methods for identifying bacteria by analysis of lipid A and/or lipoteichoic acid structure and/or mass spectrometry ionization patterns.

First claim

We claim:

1. A method for identifying bacteria to at least the genus level of classification, comprising
(a) loading a sample containing bacteria or a bacterial extract into a mass spectrometer instrument;
(b) vaporizing the sample of (a);
(c) ionizing the vaporized sample of (b) to produce precursor ions;
(d) separating the precursor ions of (c) according to the mass-to-charge ratio;
(e) detecting separated ions of (d) having values of between about 1000 m/z and about 2200 m/z;
(f) preparing precursor ion mass spectrometry (PIMS) spectra on the detected ions of (e), wherein the precursor ions are precursor ions of (i) lipid A (LA) or parent glycolipids thereof, or (ii) lipoteichoic acid (LTA) or parent glycolipids thereof, or both (i) and (ii), and wherein the PIMS spectra is PIMS spectra for (i) or (ii), or both (i) and (ii); and
(g) identifying bacteria in the sample to at least the genus level of classification by comparing the PIMS spectra prepared in (f) to a database of bacterial PIMS spectra.

2. The method of claim 1 , wherein the comparing comprises comparing precursor ion m/z values and relative abundance of the precursor ions to the database of bacterial LA and/or LTA PIMS spectra data.

3. The method of claim 1 , further comprising fragmenting all or a subset of the precursor ions to produce an MS E set of ions, and obtaining MS spectra on all or a subset of the MS E set of ions (MS E MS spectra), and wherein the comparing further comprises comparing the MS E MS spectra to bacterial LA and LTA MS E MS spectra in a database to assist in identifying bacteria in the sample.

4. The method of claim 1 , further comprising fragmenting all or a subset of the precursor ions to produce a MS n set of derived fragment ions, and obtaining MS spectra on all or a subset of the derived fragment ions (MS n MS spectra), and wherein the comparing further comprises sequentially comparing the MS n MS spectra to bacterial LA and LTA MS n MS spectra in a database to assist in identifying bacteria in the sample.

5. The method of claim 4 , further comprising searching the PIMS and/or MS n MS spectra against a database of bacterial LA and LTA signature ions to identify signature ions in the PIMS and/or MS n MS spectra.

6. The method of claim 5 , further comprising
(i) searching neutral losses of signature ions in the MS n MS spectra against a theoretical neutral loss database to identify dissociation formulae;
(ii) proposing LA and/or LTA candidate structures from bacteria in the sample based on the dissociation formulae and the signature ions in the MS n MS spectra;
(iii) assigning a score to each LA and/or LTA candidate structure based on correlation between theoretical and acquired MS n MS spectra, wherein candidate structures that meet or exceed a user-defined threshold are considered as accurate assignments.

7. The method of claim 6 , wherein step (i) comprises
(A) determining a neutral loss of every MS n MS spectrum's precursor ion in the corresponding MS n-1 MS spectrum and searching against the theoretical neutral loss database; and
(B) iteratively repeating step (A) until level MS 1 is reached; and
wherein step (ii) comprises proposing the LA and/or the LTA structures from the bacteria in the sample based on the integrating data from each MS n MS level.

8. The method of claim 6 , wherein step (iii) comprises
(A) fragmenting the LA and/or the LTA candidate structures by direct bond cleavage to produce fragmentations;
(B) combining the fragmentations into a reconstructed mass spectra representing the theoretical dissociation of the LA and/or the LTA candidate structures; and
(C) assigning the score to each of the LA and/or the LTA candidate structure based on correlation between theoretical MS n MS spectra and the reconstructed mass spectra.

9. A method for constructing libraries of LA and/or LTA precursor ion and MS E and/or MS n data, comprising
(a) loading a sample containing one or more known bacteria or an extract of one or more known bacteria into a mass spectrometer instrument;
(b) vaporizing the sample of (a);
(c) ionizing the vaporized sample of (b) to produce precursor ions;
(d) separating the precursor ions of (c) according to the mass-to-charge ratio;
(e) detecting separated ions of (d) having values of between about 1000 m/z and about 2200 m/z;
(f) preparing PIMS on the detected ions of (e), wherein the precursor ions Are precursor ions of (i) lipid A (LA) or parent glycolipids thereof, or (ii) lipoteichoic acid (LTA) or parent glycolipids thereof, or both (i) and (ii), and wherein the PIMS spectra is PIMS spectra for (i) or (ii), or both (i) and (ii);
(g) determining precursor ion m/z values and relative ratios of precursor ion signals relative to each other from the PIMS spectra of (f);
(h) determining consensus values for the precursor ion m/z values and the relative ratios of the precursor ion signals relative to each other for a given bacteria; and
(i) storing the consensus values in a database as a feature of the bacteria type.

10. The method of claim 1 , further comprising
(h) performing mass spectrometry (MS) on precursor ions for bacterial proteins in the sample, to produce protein MS spectra for the precursor ions;
(i) comparing the protein MS spectra to a database of bacterial protein PIMS spectra;
wherein the comparing is used to help identify bacteria in the sample.

Assignees

• Goodlett David R
• Ernst Robert K
• Ting Ying Sonia
• Kao Yu Chieh “Philip”
• Univ Maryland
• Univ Washington

Inventors

• Goodlett David R
• Ernst Robert K
• Ting Ying Sonia
• Kao Yu Chieh “Philip”

Classifications

• G16B10/00

  ICT specially adapted for evolutionary bioinformatics, e.g. phylogenetic tree construction or analysis · CPC title

• H01J49/004

  Combinations of spectrometers, tandem spectrometers, e.g. MS/MS, MSn · CPC title

• C12Q1/04 Primary

  Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor {(C12Q1/6897 takes precedence)} · CPC title

• G16B50/00

  ICT programming tools or database systems specially adapted for bioinformatics · CPC title

• G06F19/28

  Physics · mapped topic