3 Methods
3.1 Discovery Phase:
Sarcoplasmic Protein
Extraction
of the Selected
Seafood
In the first step, a shotgun bottom-up proteomics approach is used
to create a reference data set for the selected food (i.e., fish)
proteome (see Note 1).
1. Homogenize 5 g of the white muscle of the selected fish species
in 10 mL of lysis buffer during 30 s in an ultra-turrax device.
2. Centrifuge protein extracts at 40,000 Â g for 20 min at 4
C
and purify the supernatants into new tubes.
3. Measure protein concentration in the extracts using Lowry,
Bradford, or the bicinchoninic acid (BCA) method. The concentration is expected to be approximately 1–5 mg/mL. It is
recommended to continue with Subheading 3.1, step 2 immediately to minimize protein degradation. If sample lysates need
to be stored at this point, store them at À80
C until use.
3.2 Trypsin Protein
Digestion
1. Dissolve a total of 100 μg of protein in 8 M urea and 25 mM of
ammonium bicarbonate pH 8.0. Add DTT to a final concentration of 5 mM for 30 min at 37
C. Add IAA to a final
concentration of 50 mM and incubate for 60 min in dark.
Dilute 4Â the sample with 25 mM of ammonium bicarbonate
pH 8.25 (see Note 2).
2. Digest a total of 100 μg of protein with 1 μg of trypsin in
25 mM ammonium bicarbonate, pH 8.0 (1:100 protease to
protein) overnight at 37
C.
3. Acidify the peptide digest by the addition of 5% FA to reduce
the pH of the solution to ~2.7.
4. Clean the samples on C18 MicroSpin™ columns and dry in a
SpeedVac.
3.3 LC–MS/MS
Analysis
1. Analyze the mixture of peptides by LC–MS/MS in a Proxeon
EASY-nLC II LC system coupled to a LTQ-Orbitrap Elite
mass spectrometer (see Note 3).
2. Separate a total of 1 μg of peptides on an RP C18 column
(50 cm  75 μm, 2 μm particles, 100 A ˚ pore size) using 0.1%
FA in Milli-Q water and 0.1% FA in 98% ACN as mobile phases
A and B, respectively. Use a 60-min linear gradient from 5% to
35% B at a flow rate of 300 nL/min.
3. For the ionization step, the following parameters are required:
1.95 kV for spray voltage and 230
C for capillary temperature.
Peptides are analyzed in positive ion mode, and complete highresolution full scans are obtained from 400 to 1600 amu
(1 μscan) in the Orbitrap analyzer, followed by 10 dataShotgun Proteomics and Protein-ased Bioinformatics. . .
219
3.1 Discovery Phase:
Sarcoplasmic Protein
Extraction
of the Selected
Seafood
In the first step, a shotgun bottom-up proteomics approach is used
to create a reference data set for the selected food (i.e., fish)
proteome (see Note 1).
1. Homogenize 5 g of the white muscle of the selected fish species
in 10 mL of lysis buffer during 30 s in an ultra-turrax device.
2. Centrifuge protein extracts at 40,000 Â g for 20 min at 4
C
and purify the supernatants into new tubes.
3. Measure protein concentration in the extracts using Lowry,
Bradford, or the bicinchoninic acid (BCA) method. The concentration is expected to be approximately 1–5 mg/mL. It is
recommended to continue with Subheading 3.1, step 2 immediately to minimize protein degradation. If sample lysates need
to be stored at this point, store them at À80
C until use.
3.2 Trypsin Protein
Digestion
1. Dissolve a total of 100 μg of protein in 8 M urea and 25 mM of
ammonium bicarbonate pH 8.0. Add DTT to a final concentration of 5 mM for 30 min at 37
C. Add IAA to a final
concentration of 50 mM and incubate for 60 min in dark.
Dilute 4Â the sample with 25 mM of ammonium bicarbonate
pH 8.25 (see Note 2).
2. Digest a total of 100 μg of protein with 1 μg of trypsin in
25 mM ammonium bicarbonate, pH 8.0 (1:100 protease to
protein) overnight at 37
C.
3. Acidify the peptide digest by the addition of 5% FA to reduce
the pH of the solution to ~2.7.
4. Clean the samples on C18 MicroSpin™ columns and dry in a
SpeedVac.
3.3 LC–MS/MS
Analysis
1. Analyze the mixture of peptides by LC–MS/MS in a Proxeon
EASY-nLC II LC system coupled to a LTQ-Orbitrap Elite
mass spectrometer (see Note 3).
2. Separate a total of 1 μg of peptides on an RP C18 column
(50 cm  75 μm, 2 μm particles, 100 A ˚ pore size) using 0.1%
FA in Milli-Q water and 0.1% FA in 98% ACN as mobile phases
A and B, respectively. Use a 60-min linear gradient from 5% to
35% B at a flow rate of 300 nL/min.
3. For the ionization step, the following parameters are required:
1.95 kV for spray voltage and 230
C for capillary temperature.
Peptides are analyzed in positive ion mode, and complete highresolution full scans are obtained from 400 to 1600 amu
(1 μscan) in the Orbitrap analyzer, followed by 10 dataShotgun Proteomics and Protein-ased Bioinformatics. . .
219