3. Incubate at 37
C for 30 min and then maintain at room
temperature for 5 min. Measure the absorbance of each cuvette
in a spectrophotometer at 562 nm (see Note 10). The absorbances are compared with the calibration curve equation
obtaining the μg of protein in each sample.
3.4 Protein
Denaturalization,
Reduction, Alkylation,
and Tryptic Digestion
For sample preparation, the reduction, alkylation, fractionation,
and purification are required for the best peptide analysis.
1. A total of 100 μg of protein extract is dried under vacuum in a
Speedvac.
2. For denaturation of protein, the resulted dry extract is resuspended in 25 μl of 8 M urea in 50 mM ammonium bicarbonate
(pH 8.0) and then sonicated for 5 min.
3. The reduction is performed by the addition of 2.5 μl DTT to a
final concentration of 10 mM and then the solution was incubated at 37
C for 1 h (see Note 11).
4. Alkylation is performed with the addition of 10 μl of iodoacetamide to a final concentration of 50 mM and 22.5 μl of H 2 O
HPLC. Then the solution is incubated for 1 h at room temperature in darkness. The sample is diluted fourfold adding 400 μl
of 50 mM ammonium bicarbonate, pH 8.0, to decrease the
urea concentration (see Note 12).
5. Finally, proteins are digested with trypsin, adding 2 μl of trypsinization solution (2:100 protease-to-protein ratio) and then
incubated at 37
C overnight.
3.5 Peptide
Desalting and
Purification
1. Peptide digests are acidified by adding 5% FA until the sample is
pH 2 (see Note 13).
2. The peptide purification is performed on a C18 MicroSpin™
column. Washings of the columns were done with 200 μl of
MeOH for 10 min and then centrifuged at 100 Â g for 2 min.
Fig. 1 Graphical abstract of consecutive techniques required in food microorganism peptide profile determination for the new diagnostic peptide discovery
Proteomics for Food Microorganism Detection
209
C for 30 min and then maintain at room
temperature for 5 min. Measure the absorbance of each cuvette
in a spectrophotometer at 562 nm (see Note 10). The absorbances are compared with the calibration curve equation
obtaining the μg of protein in each sample.
3.4 Protein
Denaturalization,
Reduction, Alkylation,
and Tryptic Digestion
For sample preparation, the reduction, alkylation, fractionation,
and purification are required for the best peptide analysis.
1. A total of 100 μg of protein extract is dried under vacuum in a
Speedvac.
2. For denaturation of protein, the resulted dry extract is resuspended in 25 μl of 8 M urea in 50 mM ammonium bicarbonate
(pH 8.0) and then sonicated for 5 min.
3. The reduction is performed by the addition of 2.5 μl DTT to a
final concentration of 10 mM and then the solution was incubated at 37
C for 1 h (see Note 11).
4. Alkylation is performed with the addition of 10 μl of iodoacetamide to a final concentration of 50 mM and 22.5 μl of H 2 O
HPLC. Then the solution is incubated for 1 h at room temperature in darkness. The sample is diluted fourfold adding 400 μl
of 50 mM ammonium bicarbonate, pH 8.0, to decrease the
urea concentration (see Note 12).
5. Finally, proteins are digested with trypsin, adding 2 μl of trypsinization solution (2:100 protease-to-protein ratio) and then
incubated at 37
C overnight.
3.5 Peptide
Desalting and
Purification
1. Peptide digests are acidified by adding 5% FA until the sample is
pH 2 (see Note 13).
2. The peptide purification is performed on a C18 MicroSpin™
column. Washings of the columns were done with 200 μl of
MeOH for 10 min and then centrifuged at 100 Â g for 2 min.
Fig. 1 Graphical abstract of consecutive techniques required in food microorganism peptide profile determination for the new diagnostic peptide discovery
Proteomics for Food Microorganism Detection
209