2.3 Peptide
Preparation
1. BSA: albumin from bovine serum (0.05, 0.1, 0.2, 0.4, 0.6, 0.8,
and 1 μg/μl) in MiliQ water.
2. BCA solution: 98% of bicinchoninic acid and 2% of copper(II)
sulfate. Prepare at the time of use.
3. MiliQ water.
4. Incubator set at 31
C.
5. Speedvac.
6. 8 M urea.
7. 50 mM ammonium bicarbonate (pH 8.0).
8. Sonicator.
9. 100 mM DTT (DL-dithiothreitol) ! 98% (HPLC).
10. Incubator set at 37
C.
11. H 2 O HPLC grade.
12. 500 mM IAA (iodoacetamide).
13. Trypsinization solution: 20 μg of sequence grade modified
trypsin diluted in 20 μl of 50 mM ammonium bicarbonate.
14. 5% Formic acid (FA) grade for liquid chromatography.
15. MeOH.
16. Column Microspin C18 (25–75 ml).
17. Centrifuge.
18. 80% ACN (acetonitrile grade for liquid chromatography) and
0.1% FA (formic acid grade for liquid chromatography).
19. 2% ACN and 0.1% FA.
20. 50% ACN and 0.1% FA.
2.4 Shotgun LC–MS/
MS Analysis
1. High-resolution Q-TOF mass spectrometer (Sciex Triple TOF
5600) equipped with electrospray source for nano-flow.
2. HPLC system for nano-flow (Eksigent nanoLC415).
3. Analytical HPLC Column, C18 for nano-flow (Thermo
Acclaim PepMap C18, 25 cm long, 75 μm internal diameter,
3 μm particle size, 100 A ˚ pore size).
4. Trap column, C18 for nano-flow (Thermo Acclaim PepMap
C18, 2 cm long, 100 μm internal diameter, 5 μm particle size,
100 A ˚ pore size).
2.5 LC–MS/MS Mass
Spectrometry
Databases and
Software
1. Protein database search engine Sciex Protein Pilot v5.0.1.
2. SwissProt bacteria protein database, download it from
Uniprot.org.
3. BLASTp program against protein sequences registered in the
NCBI database.
Proteomics for Food Microorganism Detection
207
Preparation
1. BSA: albumin from bovine serum (0.05, 0.1, 0.2, 0.4, 0.6, 0.8,
and 1 μg/μl) in MiliQ water.
2. BCA solution: 98% of bicinchoninic acid and 2% of copper(II)
sulfate. Prepare at the time of use.
3. MiliQ water.
4. Incubator set at 31
C.
5. Speedvac.
6. 8 M urea.
7. 50 mM ammonium bicarbonate (pH 8.0).
8. Sonicator.
9. 100 mM DTT (DL-dithiothreitol) ! 98% (HPLC).
10. Incubator set at 37
C.
11. H 2 O HPLC grade.
12. 500 mM IAA (iodoacetamide).
13. Trypsinization solution: 20 μg of sequence grade modified
trypsin diluted in 20 μl of 50 mM ammonium bicarbonate.
14. 5% Formic acid (FA) grade for liquid chromatography.
15. MeOH.
16. Column Microspin C18 (25–75 ml).
17. Centrifuge.
18. 80% ACN (acetonitrile grade for liquid chromatography) and
0.1% FA (formic acid grade for liquid chromatography).
19. 2% ACN and 0.1% FA.
20. 50% ACN and 0.1% FA.
2.4 Shotgun LC–MS/
MS Analysis
1. High-resolution Q-TOF mass spectrometer (Sciex Triple TOF
5600) equipped with electrospray source for nano-flow.
2. HPLC system for nano-flow (Eksigent nanoLC415).
3. Analytical HPLC Column, C18 for nano-flow (Thermo
Acclaim PepMap C18, 25 cm long, 75 μm internal diameter,
3 μm particle size, 100 A ˚ pore size).
4. Trap column, C18 for nano-flow (Thermo Acclaim PepMap
C18, 2 cm long, 100 μm internal diameter, 5 μm particle size,
100 A ˚ pore size).
2.5 LC–MS/MS Mass
Spectrometry
Databases and
Software
1. Protein database search engine Sciex Protein Pilot v5.0.1.
2. SwissProt bacteria protein database, download it from
Uniprot.org.
3. BLASTp program against protein sequences registered in the
NCBI database.
Proteomics for Food Microorganism Detection
207