2.2 Protein
Concentration
1. Bicinchoninic acid (BCA) method.
2. Centrifuge.
3. Microplate Reader (Multiskan™ GO, Thermo Fisher
Scientific).
2.3 Protein Digestion
with Trypsin
1. 8 M Urea.
2. 100 mM DTT (DL-dithiothreitol) ! 98% (HPLC).
3. 500 mM IAA (iodoacetamide).
4. Sequencing grade modified trypsin (Promega, Madison,
WI, USA).
5. 25 mM ammonium bicarbonate pH 8.0.
6. Incubator set at 37
C.
7. 5% Formic acid (FA) grade for liquid chromatography.
8. SpeedVac concentrator.
9. C18 MicroSpin™ column.
2.4 Shotgun LC–MS/
MS Analysis
1. Proxeon EASY-nLC II LC system (Thermo Fisher Scientific)
coupled to a LTQ-Orbitrap Elite mass spectrometer (Thermo
Fisher Scientific).
2. Reversed phase (RP) column (EASY-Spray column,
50 cm  75 μm ID, PepMap C18, 2 μm particles, 100 A ˚
pore size) (Thermo Fisher Scientific).
3. Pre-column (Accucore XL C18, Thermo Fisher Scientific).
4. Mobile phase A: 0.1% FA in Milli-Q water.
5. Mobile phase B: 0.1% FA in 98% acetonitrile (ACN).
2.5 MS Data
Processing
1. Proteome Discoverer 2.4 package (Thermo Fisher Scientific)
implemented with SEQUEST-HT license.
2. UniProt/TrEMBL database downloaded from Uniprot.org.
2.6 Bioactive
Peptides Prediction
1. MS-Digest software included in ProteinProspector v6.2.1
website
(http://prospector.ucsf.edu/prospector/mshome.
htm).
2. PeptideRanker software (http://bioware.ucd.ie/~testing/
biowareweb/).
3. Bioactive Peptide Databases: CAMP (http://www.bicnirrh.res.
in/antimicrobial/), BIOPEP-UWM (http://www.uwm.edu.
pl/biochemia/index.php/pl/biopep/) and BioPD (http://
biopd.bjmu.edu.cn/).
218
Mo ´ nica Carrera et al.
Concentration
1. Bicinchoninic acid (BCA) method.
2. Centrifuge.
3. Microplate Reader (Multiskan™ GO, Thermo Fisher
Scientific).
2.3 Protein Digestion
with Trypsin
1. 8 M Urea.
2. 100 mM DTT (DL-dithiothreitol) ! 98% (HPLC).
3. 500 mM IAA (iodoacetamide).
4. Sequencing grade modified trypsin (Promega, Madison,
WI, USA).
5. 25 mM ammonium bicarbonate pH 8.0.
6. Incubator set at 37
C.
7. 5% Formic acid (FA) grade for liquid chromatography.
8. SpeedVac concentrator.
9. C18 MicroSpin™ column.
2.4 Shotgun LC–MS/
MS Analysis
1. Proxeon EASY-nLC II LC system (Thermo Fisher Scientific)
coupled to a LTQ-Orbitrap Elite mass spectrometer (Thermo
Fisher Scientific).
2. Reversed phase (RP) column (EASY-Spray column,
50 cm  75 μm ID, PepMap C18, 2 μm particles, 100 A ˚
pore size) (Thermo Fisher Scientific).
3. Pre-column (Accucore XL C18, Thermo Fisher Scientific).
4. Mobile phase A: 0.1% FA in Milli-Q water.
5. Mobile phase B: 0.1% FA in 98% acetonitrile (ACN).
2.5 MS Data
Processing
1. Proteome Discoverer 2.4 package (Thermo Fisher Scientific)
implemented with SEQUEST-HT license.
2. UniProt/TrEMBL database downloaded from Uniprot.org.
2.6 Bioactive
Peptides Prediction
1. MS-Digest software included in ProteinProspector v6.2.1
website
(http://prospector.ucsf.edu/prospector/mshome.
htm).
2. PeptideRanker software (http://bioware.ucd.ie/~testing/
biowareweb/).
3. Bioactive Peptide Databases: CAMP (http://www.bicnirrh.res.
in/antimicrobial/), BIOPEP-UWM (http://www.uwm.edu.
pl/biochemia/index.php/pl/biopep/) and BioPD (http://
biopd.bjmu.edu.cn/).
218
Mo ´ nica Carrera et al.