3 Methods
3.1 Preparation
of the Cell Pellet
1. Pipette 1 mL of Leptospira culture in a 1.5 mL microtube.
2. Centrifuge for 10 min at 16,000–18,000 Â g at ambient
temperature.
3. Discard EMJH supernatant and resuspend leptospires in
200 μL PBS by pipetting.
4. Centrifuge for 10 min at 16,000–18,000 Â g at ambient
temperature.
5. Discard supernatant and resuspend pellet with 300 μL ultrapure water by pipetting and vortexing if needed. A homogenous cloudy suspension must be obtained.
6. Add 900 μL absolute ethanol and homogenize by pipetting
and/or vortexing (see Note 3).
7. Centrifuge for 10 min at 16,000–18,000 Â g at ambient
temperature.
8. Discard the supernatant and allow the pellet to dry completely
by opening the tube lid under the biosafety cabinet (see Note
4).
9. Add 10–20-μL formic acid depending on the size of the pellet
(see Note 5). Vigorously homogenize by pipetting up and
down multiple times.
10. Add the same volume acetonitrile and homogenize by pipetting up and down.
11. Centrifuge for 2 min at 15,000 Â g at ambient temperature.
12. Spot 1 μL of this preparation on a spot of the MALDI-ToF
target plate (see Note 6).
13. Dry at ambient temperature.
14. Within 10 min after complete drying, add 1.2 μL HCCA
matrix and dry at ambient temperature.
15. Place the target plate into the MALDI-ToF.
3.2 Creation of a
Reference Spectrum
(Main Spectral
Projection, MSP)
To create a reference spectrum, a minimum of eight spots of the
strain to be analyzed must be prepared. Each of these eight spots
must then be analyzed three times, leading to a total of 24 individual mass spectra, which will be used to create the reference spectrum of the strain, named “MSP” for main spectral projection (see
Note 7). Individual spectra are normalized and compared visually.
Atypical or poor-quality spectra can be excluded from the complete
set, but at least 20 individual spectral acquisitions must be kept to
create the reference MSP. Figure 1 shows the MSP-derived phylogeny of 35 Leptospira species.
Leptospira Identification Using MALDI-ToF
25
3.1 Preparation
of the Cell Pellet
1. Pipette 1 mL of Leptospira culture in a 1.5 mL microtube.
2. Centrifuge for 10 min at 16,000–18,000 Â g at ambient
temperature.
3. Discard EMJH supernatant and resuspend leptospires in
200 μL PBS by pipetting.
4. Centrifuge for 10 min at 16,000–18,000 Â g at ambient
temperature.
5. Discard supernatant and resuspend pellet with 300 μL ultrapure water by pipetting and vortexing if needed. A homogenous cloudy suspension must be obtained.
6. Add 900 μL absolute ethanol and homogenize by pipetting
and/or vortexing (see Note 3).
7. Centrifuge for 10 min at 16,000–18,000 Â g at ambient
temperature.
8. Discard the supernatant and allow the pellet to dry completely
by opening the tube lid under the biosafety cabinet (see Note
4).
9. Add 10–20-μL formic acid depending on the size of the pellet
(see Note 5). Vigorously homogenize by pipetting up and
down multiple times.
10. Add the same volume acetonitrile and homogenize by pipetting up and down.
11. Centrifuge for 2 min at 15,000 Â g at ambient temperature.
12. Spot 1 μL of this preparation on a spot of the MALDI-ToF
target plate (see Note 6).
13. Dry at ambient temperature.
14. Within 10 min after complete drying, add 1.2 μL HCCA
matrix and dry at ambient temperature.
15. Place the target plate into the MALDI-ToF.
3.2 Creation of a
Reference Spectrum
(Main Spectral
Projection, MSP)
To create a reference spectrum, a minimum of eight spots of the
strain to be analyzed must be prepared. Each of these eight spots
must then be analyzed three times, leading to a total of 24 individual mass spectra, which will be used to create the reference spectrum of the strain, named “MSP” for main spectral projection (see
Note 7). Individual spectra are normalized and compared visually.
Atypical or poor-quality spectra can be excluded from the complete
set, but at least 20 individual spectral acquisitions must be kept to
create the reference MSP. Figure 1 shows the MSP-derived phylogeny of 35 Leptospira species.
Leptospira Identification Using MALDI-ToF
25