type, identification; cys alkylation, iodoacetamide; digestion,
trypsin; instrument, TripleTOF 5600; ID focus, biological
modifications; search effort, thorough ID.
2. From the resulting identified proteins and peptides, take only
those with a false discovery rate (FDR) below 1%.
3.8 Selection of
Potential Peptide
Biomarkers
1. For each peptide identified by LC–ESI-MS/MS, BLASTp program is used to determine homologies and exclusiveness with
protein sequences registered in the NCBI database [17] (see
Note 18).
4 Notes
1. The bacterial growth at 31
C was standardized for the analysis
of specific strains isolated from food by mass spectrometry. In
general, bacterial strains with optimal growth temperatures at
37
C grow well at 31
C.
2. Tubes of broth are inoculated under aerobic conditions.
3. Each strain analysis is performed in triplicate.
4. Depending on the solubility of protein required for extraction,
different compatible solvents are used (a,b,c).
5. This step could be avoided in case target proteins are only
membrane proteins.
6. After each pulse in the dismembrator, the sample must be
placed on ice, avoiding the increase of temperature. The dismembrator is used before the addiction of detergents to avoid
foam formation during this step.
7. Different compatible detergents (such as a and b) can be used
in buffer lysis solution at the final step of protein extraction.
8. The sample must be dry before the next step. If the sample is
not entirely dry after 4 h in the speed vacuum, it must be left for
a few additional minutes until it is dried.
9. The quantification of each sample protein may be doubly
measured. The blank is prepared with MiliQ water.
10. When the measured absorbance of a sample exceeds the calibration curve, the volume of sample is reduced by adding
MiliQ water to a final volume of 50 μl and recalculating the
μg of protein compared to the calibration curve.
11. DTT (DL-dithiothreitol) is used to stabilize proteins, to reduce
disulfide bonds and to maintain monothiols in a reduced state.
12. Iodoacetamide is a sulfhydryl-reactive alkylating reagent used
to bind covalently with the thiol group of cysteines in order to
avoid disulfide bond formation for protein characterization
and peptide mapping.
Proteomics for Food Microorganism Detection
211
trypsin; instrument, TripleTOF 5600; ID focus, biological
modifications; search effort, thorough ID.
2. From the resulting identified proteins and peptides, take only
those with a false discovery rate (FDR) below 1%.
3.8 Selection of
Potential Peptide
Biomarkers
1. For each peptide identified by LC–ESI-MS/MS, BLASTp program is used to determine homologies and exclusiveness with
protein sequences registered in the NCBI database [17] (see
Note 18).
4 Notes
1. The bacterial growth at 31
C was standardized for the analysis
of specific strains isolated from food by mass spectrometry. In
general, bacterial strains with optimal growth temperatures at
37
C grow well at 31
C.
2. Tubes of broth are inoculated under aerobic conditions.
3. Each strain analysis is performed in triplicate.
4. Depending on the solubility of protein required for extraction,
different compatible solvents are used (a,b,c).
5. This step could be avoided in case target proteins are only
membrane proteins.
6. After each pulse in the dismembrator, the sample must be
placed on ice, avoiding the increase of temperature. The dismembrator is used before the addiction of detergents to avoid
foam formation during this step.
7. Different compatible detergents (such as a and b) can be used
in buffer lysis solution at the final step of protein extraction.
8. The sample must be dry before the next step. If the sample is
not entirely dry after 4 h in the speed vacuum, it must be left for
a few additional minutes until it is dried.
9. The quantification of each sample protein may be doubly
measured. The blank is prepared with MiliQ water.
10. When the measured absorbance of a sample exceeds the calibration curve, the volume of sample is reduced by adding
MiliQ water to a final volume of 50 μl and recalculating the
μg of protein compared to the calibration curve.
11. DTT (DL-dithiothreitol) is used to stabilize proteins, to reduce
disulfide bonds and to maintain monothiols in a reduced state.
12. Iodoacetamide is a sulfhydryl-reactive alkylating reagent used
to bind covalently with the thiol group of cysteines in order to
avoid disulfide bond formation for protein characterization
and peptide mapping.
Proteomics for Food Microorganism Detection
211