3 Method
All consecutive steps (Fig. 1) of this protocol are used for the food
microorganism peptide profile determination and consequently
diagnosis peptide discovery for food microorganism detection.
3.1 Strain Culture
1. Reactivate the bacterial strains in BHI medium and incubate
them at 31
C for 24 h.
2. Bacterial cultures are then grown on PCA plates at 31
C (see
Note 1) for 24 h (see Note 2).
3.2 Protein
Extraction
Following the bacterial culture, protein extraction is performed and
specific proteins analyzed. Different lysate preparation and even
physical-based method may be resquired to extract cellular
proteins.
1. Protein extraction is prepared harvesting a loop full of bacterial
culture (see Note 3).
2. The biomass can be mixed with either (a) 100 μl of a solution
containing 50% ACN and 1% TFA, (b) 200 μl of a solution of
75 mM NaCl and 50 mM Tris–HCl, or (c) 200 μl 80% TFA (see
Note 4).
3. Dismembrator is used in the next step (see Note 5) for soluble
protein extraction. It is performed by applying 3 pulses for 30 s,
adding the same biomass volume of 0.1 mm glass beads (see
Note 6).
4. The sample is then centrifugated for 10 min at 6000 Â g. After
centrifugate the mixture, the supernatant is treated with a
solution of lysis buffer: (a) 200 μl lauryl maltoside buffer lysis
solution or (b) 200 μl of n-docecyl-D-maltoside buffer lysis
solution; and is resuspended by vortexing (see Note 7).
5. The samples are dried in a speed vacuum for 4 h (see Note 8).
3.3 Soluble Protein
Quantification
For the protein digestion, previous protein quantification is
required. The measurement of protein is performed by the bicinchoninic acid (BCA) method and the construction of a calibration
curve.
1. For the calibration curve, prepare tubes with different concentrations of BSA (0.05, 0.1, 0.2, 0.4, 0.6, 0.8, and 1 μg/μl),
adding MiliQ water until a final volume of 500 μl per tube.
Take 50 μl of each BSA solution adding 1 ml of BCA to a
spectrometer cuvette.
2. For the measurement of the sample protein, take 50 μl of each
sample, adding 1 ml of BCA to a spectrometer cuvette (see
Note 9).
208
Ana G. Abril et al.
All consecutive steps (Fig. 1) of this protocol are used for the food
microorganism peptide profile determination and consequently
diagnosis peptide discovery for food microorganism detection.
3.1 Strain Culture
1. Reactivate the bacterial strains in BHI medium and incubate
them at 31
C for 24 h.
2. Bacterial cultures are then grown on PCA plates at 31
C (see
Note 1) for 24 h (see Note 2).
3.2 Protein
Extraction
Following the bacterial culture, protein extraction is performed and
specific proteins analyzed. Different lysate preparation and even
physical-based method may be resquired to extract cellular
proteins.
1. Protein extraction is prepared harvesting a loop full of bacterial
culture (see Note 3).
2. The biomass can be mixed with either (a) 100 μl of a solution
containing 50% ACN and 1% TFA, (b) 200 μl of a solution of
75 mM NaCl and 50 mM Tris–HCl, or (c) 200 μl 80% TFA (see
Note 4).
3. Dismembrator is used in the next step (see Note 5) for soluble
protein extraction. It is performed by applying 3 pulses for 30 s,
adding the same biomass volume of 0.1 mm glass beads (see
Note 6).
4. The sample is then centrifugated for 10 min at 6000 Â g. After
centrifugate the mixture, the supernatant is treated with a
solution of lysis buffer: (a) 200 μl lauryl maltoside buffer lysis
solution or (b) 200 μl of n-docecyl-D-maltoside buffer lysis
solution; and is resuspended by vortexing (see Note 7).
5. The samples are dried in a speed vacuum for 4 h (see Note 8).
3.3 Soluble Protein
Quantification
For the protein digestion, previous protein quantification is
required. The measurement of protein is performed by the bicinchoninic acid (BCA) method and the construction of a calibration
curve.
1. For the calibration curve, prepare tubes with different concentrations of BSA (0.05, 0.1, 0.2, 0.4, 0.6, 0.8, and 1 μg/μl),
adding MiliQ water until a final volume of 500 μl per tube.
Take 50 μl of each BSA solution adding 1 ml of BCA to a
spectrometer cuvette.
2. For the measurement of the sample protein, take 50 μl of each
sample, adding 1 ml of BCA to a spectrometer cuvette (see
Note 9).
208
Ana G. Abril et al.