Analysis of microbes from microcolonies by maldi mass spectrometry
View Patent ↗The invention relates to the cell disruption of microbes and the preparation of the microbe proteins for mass spectrometric analysis. The cells of microbes from microcolonies are disrupted by physical or chemical means directly on the nutrient medium. The released proteins are then transferred to sample supports by direct contact with their contact surfaces; electrophoresis can be used for assistance. Once the proteins are firmly adsorbed on the contact surfaces, they can be washed with water in order to remove substances which interfere with the ionization process. For analysis by matrix-assisted laser desorption (MALDI), the proteins are prepared on the contact surfaces of the sample supports with matrix substances to form MALDI samples; the sample supports are then introduced into a MALDI mass spectrometer for the acquisition of mass spectra. The microbes are identified by similarity comparisons between the mass spectra of the microbe proteins and similarly obtained reference spectra.
1. A method for the mass spectrometric analysis of microbes on the surface of a nutrient medium in a mass spectrometer with ionization by matrix-assisted laser desorption, comprising the steps:
(a) disrupting the cells of microbes on the surface of the nutrient medium,
(b) transferring the microbe proteins released by the cell disruption onto a contact surface of a sample support by direct contact of the contact surface with the cell disrupted microbes,
(c) preparing the transferred microbe proteins as a MALDI sample on the contact surface of the sample support, and
(d) transferring the sample support with the MALDI sample to the mass spectrometer.
2. A method according to claim 1 , wherein the end surface of a pin-shaped sample support is brought into contact with the cell disrupted microbes, the contact surface of the pin-shaped sample support being so small that only microbe proteins of an individual colony are transferred onto the pin-shaped sample support.
3. A method according to claim 2 , wherein, after the microbe proteins have been transferred, the pin-shaped sample support is inserted into an adapter plate in such a way that the end surface of the pin-shaped sample support is essentially flush with the surface of the adapter plate with a positive fit.
4. A method according to claim 3 , with the steps:
taking an image of the surface of the nutrient medium,
determining the positions of colonies from the image,
disrupting the microbes at the determined positions,
transferring the cell disrupted microbe proteins at the determined positions onto a separate pin-shaped sample support,
inserting the pin-shaped sample supports into an adapter plate,
preparing MALDI samples from the microbe proteins on the pin-shaped sample supports,
introducing the adapter plate into a mass spectrometer, and acquiring spectra with ionization by matrix-assisted laser desorption at the positions of the pin-shaped sample supports in the adapter plate.
5. A method according to one of the claim 4 , wherein the end surfaces of the pin-shaped sample supports have surfaces of less than nine square millimeters, preferably less than four square millimeters.
6. A method according to claim 1 , wherein a plate-shaped sample support is brought into contact with the cell disrupted microbes, and the contact surface of the plate-shaped sample support is so large that microbe proteins from microbes of several colonies are transferred simultaneously onto the plate-shaped sample support.
7. A method according to claim 6 , wherein the surface of the nutrient medium is imaged before or after cell disruption of the microbes, the position of the plate-shaped sample support relative to the surface of the nutrient medium during the contact transfer is determined, and the positions of the microbe proteins on the sample support are determined from the image and the position of the sample support plate.
8. A method according to claim 7 , wherein the MALDI samples are prepared only at the determined positions, and the mass spectrometric analyses are carried out only on the prepared MALDI samples, or a matrix layer is prepared on the whole of the contact surface of the sample support and the mass spectrometric analyses are carried out only at the determined positions.
9. A method according to claim 6 , wherein, during inoculation of the sample for analysis onto the surface of the nutrient medium, the track of the inoculation is recorded and the mass spectrometric analyses are carried out only along the recorded track.
10. A method according to claim 1 , wherein the microbes are cultured on the surface of the nutrient medium for less than eight hours before being cell disrupted.
11. A method according to claim 1 , wherein the cells of the microbes are disrupted physically on the surface of the nutrient medium by ultrasound, infrared radiation or high pressure.
12. A method according to claim 11 , wherein the positions of colonies on the surface of the nutrient medium are determined, colonies are selected, and the ultrasound or the infrared radiation has a localized effect on the colonies selected.
13. A method according to claim 1 , wherein the cells of the microbes are disrupted chemically by adding substance solutions.
14. A method according to claim 13 , wherein the positions of colonies on the surface of the nutrient medium are determined, colonies are selected, and the cell disrupting substance is added in a localized way to each of the selected colonies.
15. A method according to claim 13 , wherein the substance is an acid or the enzyme lysozyme.
16. A method according to claim 1 , wherein the contact surface of the sample support has a protein-adsorptive coating.
17. A method according to claim 16 , wherein the protein-adsorptive coating consists of nitrocellulose or α-cyano-4-hydroxycinnamic acid (HCCA).
18. A method according to claim 17 , wherein the microbe proteins are drawn onto the protein-adsorptive coatings by electrophoresis.
19. A method for the mass spectrometric analysis of microbes on the surface of a nutrient medium by mass spectra of their proteins acquired in a mass spectrometer, comprising the steps:
(a) disrupting the cells of microbes on the surface of a nutrient medium, thereby releasing microbe proteins,
(b) transferring the microbe proteins released onto a contact surface of a sample support by direct contact, and
(c) acquiring mass spectra of the microbial proteins on the sample support.