6. SDS-PAGE running buffer: 0.5 M Tris–HCl, pH 8.3, 0.192 M
glycine, 0.1% SDS (see Note 7).
7. Bromophenol blue (BPB) solution: dissolve 0.1 g BPB in
100 mL water.
8. Loading buffer: 0.28 mL of 0.5 M Tris–HCl (pH 6.8),
0.44 mL of 10% SDS, 6.32 mg of DTT, 0.06 mL of 1%
bromophenol blue, 0.22 mL of 45% glycerol. Leave one aliquot at 4
C for current use and store the remaining aliquots at
À20
C (see Note 8).
9. Protein molecular weight marker.
10. Silver stain system for protein staining in polyacrylamide gels.
11. Fix solution: mix 60% water, 30% ethanol, 10% acetic acid.
2.5 Protein Digestion
1. 1 M triethylammonium bicarbonate (TEAB) buffer.
2. 100 mM TEAB: add 500 μL of the 1 M TEAB to 4.5 mL of
water.
3. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) stock
solution: 14 mg of TCEP dissolve in 100 mL of water.
4. 200 mM TCEP buffer: add 70 μL of TCEP stock solution to
70 μL of water. Then add 35 μL of 1 M TEAB.
5. 375 mM iodoacetamide (IAA): prepare immediately before
use, dissolve 5 mg of IAA in 74 μL of 100 mM TEAB. Protect
the solution from light.
6. Sequencing grade modified trypsin: 2 μg of trypsin per 100 μg
of protein.
2.6 TMT Labeling
1. TMT 10plex labeling set indicated for ten samples (see Note 9).
2. Anhydrous acetonitrile (ACN).
3. 5% Hydroxylamine: mix 10 μL of the 50% hydroxylamine with
10 μL of 1 M TEAB and 80 μL of water.
2.7 Peptide
Fractionation
and Cleanup
1. High pH reversed-phase peptide fractionation kit.
2. 0.1% Trifluoroacetic acid (TFA): add 10 μL of TFA to 10 mL of
water.
3. SepPak C18 cartridge.
4. Quantitative colorimetric peptide assay.
2.8 LC–MS/MS
Analysis
1. Reversed-phase (RP) column (EASY-Spray column,
50 cm  75 μm ID, PepMap C18, 2 μm particles, 100 A ˚
pore size).
2. Pre-column (Accucore XL C18).
3. Mobile phase A: 0.1% formic acid (FA) in Milli-Q water.
4. Mobile phase B: 0.1% FA in 98% ACN.
Shotgun Proteomics for Anisakis simplex
63
glycine, 0.1% SDS (see Note 7).
7. Bromophenol blue (BPB) solution: dissolve 0.1 g BPB in
100 mL water.
8. Loading buffer: 0.28 mL of 0.5 M Tris–HCl (pH 6.8),
0.44 mL of 10% SDS, 6.32 mg of DTT, 0.06 mL of 1%
bromophenol blue, 0.22 mL of 45% glycerol. Leave one aliquot at 4
C for current use and store the remaining aliquots at
À20
C (see Note 8).
9. Protein molecular weight marker.
10. Silver stain system for protein staining in polyacrylamide gels.
11. Fix solution: mix 60% water, 30% ethanol, 10% acetic acid.
2.5 Protein Digestion
1. 1 M triethylammonium bicarbonate (TEAB) buffer.
2. 100 mM TEAB: add 500 μL of the 1 M TEAB to 4.5 mL of
water.
3. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) stock
solution: 14 mg of TCEP dissolve in 100 mL of water.
4. 200 mM TCEP buffer: add 70 μL of TCEP stock solution to
70 μL of water. Then add 35 μL of 1 M TEAB.
5. 375 mM iodoacetamide (IAA): prepare immediately before
use, dissolve 5 mg of IAA in 74 μL of 100 mM TEAB. Protect
the solution from light.
6. Sequencing grade modified trypsin: 2 μg of trypsin per 100 μg
of protein.
2.6 TMT Labeling
1. TMT 10plex labeling set indicated for ten samples (see Note 9).
2. Anhydrous acetonitrile (ACN).
3. 5% Hydroxylamine: mix 10 μL of the 50% hydroxylamine with
10 μL of 1 M TEAB and 80 μL of water.
2.7 Peptide
Fractionation
and Cleanup
1. High pH reversed-phase peptide fractionation kit.
2. 0.1% Trifluoroacetic acid (TFA): add 10 μL of TFA to 10 mL of
water.
3. SepPak C18 cartridge.
4. Quantitative colorimetric peptide assay.
2.8 LC–MS/MS
Analysis
1. Reversed-phase (RP) column (EASY-Spray column,
50 cm  75 μm ID, PepMap C18, 2 μm particles, 100 A ˚
pore size).
2. Pre-column (Accucore XL C18).
3. Mobile phase A: 0.1% formic acid (FA) in Milli-Q water.
4. Mobile phase B: 0.1% FA in 98% ACN.
Shotgun Proteomics for Anisakis simplex
63