Rapid identification of bacteria by mass spectrometry

Details Rapid identification of bacteria by mass spectrometry Rapid identification of bacteria by mass spectrometry

1999-02-08

2001-01-23

Graser, Jennifer (Department: 1641)

Chemistry: molecular biology and microbiology

Treatment of micro-organisms or enzymes with electrical or...

C435S173400, C436S173000, C436S518000, C250S281000, C250S282000, C250S287000, C313S564000

Reexamination Certificate

active

06177266

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to the rapid identification of bacteria in environmental and biological samples. More specifically, the present invention relates to a method for the chemotaxonomic classification of bacteria with genus, species and strain specific biomarkers generated by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of either cellular protein extracts or whole cells.
BACKGROUND OF THE INVENTION
Rapid and accurate microbial identification is critical in diagnosing diseases, predicting on-coming public health hazards, monitoring potential contamination in stored foods and grains, regulating bioprocessing operations and recognizing warfare threats. It is important not only to rapidly distinguish between related organisms but also to unambiguously identify species and strains in complex matrices for risk assessment in field situations.
The classification of micro-organisms has traditionally been based on biochemical and morphological culturing tests. Recently, several instrumental analytical techniques have been developed which enhance the speed and accuracy of identification of bacteria cells. In these techniques, the biochemical components of bacteria cells are examined to determine chemotaxonomic markers which are specific for each bacteria species. The chemotaxonomic markers, or biomarkers, may be any one or a combination of the classes of molecules present in the cells such as lipids, phospholipids, lipopolysaccharides, oligosaccharides, proteins and DNA.
For example, a commercial microbial identification system uses gas chromatographic analysis of fatty acid methyl esters (Microbial Identification, Inc., Newark, Del.). Chemotaxonomic identification of bacteria based on fatty acid or whole-cell pyrolysis mass spectra and fast-atom bombardment mass spectrometric analysis of phospholipids has also been reported. These techniques analyze primarily the lower molecular weight lipids of the cell.
Bacteria may also be differentiated on the basis of cellular protein content. Since the proteins found in bacteria provide indirect genetic information on the organism and are related to bacterial virulence, protein content is specific to individual strains. The most established technique for examining cellular protein content is sodium deodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) which produces characteristic migration patterns for different bacteria species. Identification of a bacteria producing a particular migration pattern is accomplished by computerized comparison with reference gel patterns. However, because SDS-PAGE analysis is slow, labor intensive and requires fairly large amounts of sample material, it is not particularly useful for rapid identification of bacteria, particularly in field situations.
A limited number of recent reports have investigated the applicability of various mass spectrometric techniques for generating bacteria specific protein profiles. These techniques generally employ electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI) of bacteria protein extracts followed by mass spectrometric (MS) or tandem mass spectrometric (MS/MS) analysis. For example, the MALDI technique, combined with time-of-flight mass spectrometry (TOF-MS), has been used to differentiate bacteria using a crude protein extract requiring minimal sample preparation. (See T. C. Cain, D. M. Lubman, and W. J. Weber, Jr.,
Rapid Commun. Mass Spectrom., Vol.
8, pp. 1026-1030 (1994)). In addition, two different groups have reported the identification of intact bacteria with MALDI-TOF-MS. (See R. D. Holland et al.,
Rapid Communications in Mass Spectrometry
, Vol. 10, pp. 1227-1232 (1996) and M. A. Claydon et al.,
Nature Biotechnology
, Vol. 14, pp. 1584-1586).
The MALDI-MS technique is based on the discovery in the late 1980s that desorption/ionization of large, nonvolatile molecules such as proteins can be effected when a sample of such molecules is irradiated after being codeposited with a large molar excess of an energy-absorbing “matrix” material, even though the molecule does not strongly absorb at the wavelength of the laser radiation. The abrupt energy absorption initiates a phase change in a microvolume of the absorbing sample from a solid to a gas while also inducing ionization of the sample molecules.
Detailed descriptions of the MALDI-TOF-MS technique and its applications may be found in review articles by E. J. Zaluzec et al. (
Protein Expression and Purification
, Vol. 6, pp. 109-123 (1995)) and D. J. Harvey (
Journal of Chromatography A
, Vol. 720, pp. 429-4446 (1996)), each of which is incorporated herein by reference. In brief, the matrix and analyte are mixed to produce a solution with a matrix:analyte molar ratio of approximately 10,000:1. A small volume of this solution, typically 0.5-2 &mgr;l, is applied to a stainless steel probe tip and allowed to dry. During the drying process the matrix codeposits from solution with the analyte.
Ionization of the analyte is effected by pulsed laser radiation focused onto the probe tip which is located in a short (~5 cm) source region containing an electric field. The ions formed at the probe tip are accelerated by the electric field toward a detector through a flight tube, which is a long (~1 m) field free drift region. Since all ions receive the same amount of energy, the time required for ions to travel the length of the flight tube is dependent on their mass. Thus, low-mass ions have a shorter time of flight (TOF) than heavier ions. All the ions that reach the detector as the result of a single laser pulse produce a transient TOF signal. Typically, ten to several hundred transient TOF mass spectra are averaged to improve ion counting statistics.
The mass of an unknown analyte is determined by comparing its experimentally determined TOF to TOF signals obtained with ions of known mass. The MALDI-TOF-MS technique is capable of determining the mass of proteins of between 1 and 40 kDa with a typical accuracy of ±0.1%, and a somewhat lower accuracy for proteins of molecular mass above 40 kDa.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide an improved mass spectrometric method for identifying bacteria at the genus, species and strain levels.
Another object of the present invention is to provide such a method which may be applied to whole bacteria cells in the laboratory or in field situations.
Yet another object of the present invention is to provide a library of genus, species and strain specific biomarkers for use in bacteria identification by mass spectrometry.
DETAILED DESCRIPTION OF THE INVENTION
These and other objects are satisfied by the present invention which includes a method for generating unique mass spectral profiles, as well as individual biomarkers, of the proteinaceous material in bacteria extracts or whole bacteria cells by MALDI-TOF-MS, and the specific biomarkers produced by the method. The method has been described in detail elsewhere. See T. Krishnamurthy, et al.,
Rapid Communications in Mass Spectrometry
, Vol 10, pp. 883-888 (1996). T. Krishnamurthy and P. Ross,
Rapid Communications in Mass Spectrometry
, Vol 10, pp. 1992-96 (1996), and T. Krishnamurthy and P. Ross,
Proceedings of Seventh National Symposium on Mass Spectrometry
, Indian Society for Mass Spectrometry, pp. 105-122 (November 1996), each of which is incorporated herein by reference.
A sample mixture is prepared by mixing a matrix solution with an unknown bacteria sample which comprises a protein extract or whole cells. A small aliquot 1 &mgr;l of the sample mixture is subjected to MS analysis in a MALDI-TOF instrument equipped with a nitrogen laser. Mass spectra are averaged over 100-200 individual laser shots, collected, and compared to MALDI-TOF-MS spectra of protein extracts or whole cells of known bacteria to identify the unknown bacteria sample.
As stated above, the present invention provides a method for generating unique mass spectral profiles for bacteria protei

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