3.3 Acetone–
Methanol Protein
Precipitation
and Extraction
1. Precipitate proteins by adding 9 ml acetone/methanol (8:1)
and incubate overnight at À20
.
2. Centrifuge samples at 4000 Â g for 10 min.
3. Wash pellet with 80% acetone and centrifuge at 4000 Â g for
10 min.
4. Briefly air dry the protein pellet.
5. Resuspend the pellet in denaturation buffer.
6. Determine protein quantification using Bradford assay.
3.4 Reduction,
Alkylation,
Predigestion (See
Note 5)
1. Reduce protein sample(s) with 3 mM dithiothreitol (DTT) for
20 min.
2. Alkylate protein samples(s) with 16.5 mM iodoacetamide
(IAA) for 20 min in the dark.
3. Predigest protein sample(s) with Lys-C for 3 h at 30
C (see
Note 6).
3.5 Tryptic Digestion
1. Dilute samples with 6 volumes of 50 mM ammonium bicarbonate (see Note 6).
2. Digest samples with trypsin (1:75, Promega) with gentle shaking overnight at 30
C.
3. To stop digestion, add FA to pH 2.
3.6 StageTip
Desalting
1. StageTip protocol used was adapted from Rappsilber et al [6].
2. Activate the C18 in the stage tips by adding 100μl solvent
B. Centrifuge at 1000 Â g for 1 min to pass the liquid through
the tip into the Eppendorf tube (see Note 7a).
3. Equilibrate the C18 with 200 μL solvent A, spin at 3000 Â g
for 1 min (see Note 7b).
4. Add 10μg of acidified sample to each stage tip and centrifuge at
500 Â g, until sample flows through (see Note 7c).
5. Wash with 200 μL solvent A, spin at 3000 Â g for 1 min (see
Note 7d).
6. Insert a glass insert into the Eppendorf tube and then replace
the stage tip so that it fits into the glass insert.
7. Elute the peptides with 100 μL solvent C into the glass insert,
slow spin of 500 Â g, until sample flows through (see Note 7e).
3.7 Preparing
Peptides for MS
Analysis
1. Dry the eluted peptides in a speed-vac.
2. Store in À20
C freezer until ready to use.
3. Suspend peptides at a final concentration of 1μg/μL in
solvent A.
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John Iradukunda et al.
Methanol Protein
Precipitation
and Extraction
1. Precipitate proteins by adding 9 ml acetone/methanol (8:1)
and incubate overnight at À20
.
2. Centrifuge samples at 4000 Â g for 10 min.
3. Wash pellet with 80% acetone and centrifuge at 4000 Â g for
10 min.
4. Briefly air dry the protein pellet.
5. Resuspend the pellet in denaturation buffer.
6. Determine protein quantification using Bradford assay.
3.4 Reduction,
Alkylation,
Predigestion (See
Note 5)
1. Reduce protein sample(s) with 3 mM dithiothreitol (DTT) for
20 min.
2. Alkylate protein samples(s) with 16.5 mM iodoacetamide
(IAA) for 20 min in the dark.
3. Predigest protein sample(s) with Lys-C for 3 h at 30
C (see
Note 6).
3.5 Tryptic Digestion
1. Dilute samples with 6 volumes of 50 mM ammonium bicarbonate (see Note 6).
2. Digest samples with trypsin (1:75, Promega) with gentle shaking overnight at 30
C.
3. To stop digestion, add FA to pH 2.
3.6 StageTip
Desalting
1. StageTip protocol used was adapted from Rappsilber et al [6].
2. Activate the C18 in the stage tips by adding 100μl solvent
B. Centrifuge at 1000 Â g for 1 min to pass the liquid through
the tip into the Eppendorf tube (see Note 7a).
3. Equilibrate the C18 with 200 μL solvent A, spin at 3000 Â g
for 1 min (see Note 7b).
4. Add 10μg of acidified sample to each stage tip and centrifuge at
500 Â g, until sample flows through (see Note 7c).
5. Wash with 200 μL solvent A, spin at 3000 Â g for 1 min (see
Note 7d).
6. Insert a glass insert into the Eppendorf tube and then replace
the stage tip so that it fits into the glass insert.
7. Elute the peptides with 100 μL solvent C into the glass insert,
slow spin of 500 Â g, until sample flows through (see Note 7e).
3.7 Preparing
Peptides for MS
Analysis
1. Dry the eluted peptides in a speed-vac.
2. Store in À20
C freezer until ready to use.
3. Suspend peptides at a final concentration of 1μg/μL in
solvent A.
184
John Iradukunda et al.