US 9,803,229 B2 · Method of detection of gram-negative bacteria periplasmic space and cell wall outer membrane proteins by mass spectrometry

Abstract

The invention relates to a method of detection of Gram-negative bacteria periplasmic space and cell wall outer membrane proteins by mass spectrometry, wherein the periplasmic space and cell wall outer membrane proteins are extracted from the bacteria, and the proteins to be detected are stabilized by an inhibitor and/or a substrate of the given protein, the proteins are then dissolved, placed onto a MALDI-TOF plate, covered with matrix solution, measured by MALDI-TOF mass spectrometry, and the resulting spectra are compared to the reference peaks of the given protein. Preferably, the proteins are beta-lactamases and their detection can be used to quickly determine the bacterial resistance against beta-lactam antibiotics, minimizing the false-positive results.

Claims10

Dependent claims

A method of detection of gram-negative bacteria periplasmic space β-lactamases by MALDI-TOF mass spectrometry, the method comprising:

(a) obtaining β-lactamases from gram-negative bacteria;
(b) contacting the β-lactamases with an effective amount of an inhibitor of and/or a substrate of the β-lactamases so as to stabilize the β-lactamases;
(c) dissolving the stabilized β-lactamases;
(d) placing suitable amounts of the dissolved β-lactamases onto a MALDI-TOF plate and coating the placed β-lactamases with a matrix solution;
(e) measuring the spectra of the β-lactamases from step (d) by MALDI-TOF mass spectrometry;
and
(f) comparing the measured spectra from step (e) and/or peaks thereof to one or more reference
β-lactamases spectra and/or peaks thereof, thereby detecting the periplasmic space β-lactamase.

5 more elements

A method of detecting gram-negative bacteria periplasmic space β-lactamases by MALDI-TOF mass spectrometry, the method comprising:

(a) obtaining β-lactamases from gram-negative bacteria;
(b) contacting the β-lactamases with an effective amount of an inhibitor of and/or a substrate of the β-lactamases so as to stabilize the β-lactamases;
(c) dissolving the stabilized β-lactamases,
(d) precipitating the dissolved β-lactamases,
(e) dissolving the precipitated β-lactamases,
(f) placing suitable amounts of the dissolved β-lactamases from step (e) onto a MALDI-TOF plate and coating the placed β-lactamases with a matrix solution, wherein the matrix solution is a solution suitable for ionization of proteins with molecular mass greater than 10,000 Da,
(g) measuring the spectra of the β-lactamases from step (f) by MALDI-TOF mass spectrometry;
and
(h) comparing the measured spectra from step (g) and/or peaks thereof to one or more reference β-lactamase spectra and/or peaks thereof, thereby detecting the periplasmic space β-lactamases.

6 more elements

Dependencies are inferred from the claim text.

Method of detection of gram-negative bacteria periplasmic space and cell wall outer membrane proteins by mass spectrometry

Life of this patent

Method of detection of gram-negative bacteria periplasmic space and cell wall outer membrane proteins by mass spectrometry

Life of this patent

Abstract

Claims10