2. Buffer A: 0.1% FA in double distilled water (ddH 2 O).
3. Buffer B: 0.1% FA in 90% ACN.
3 Methods
3.1 Protein
Extraction
and Quantification
1. Add approximately 200 μL of RIPA buffer (see Note 4) with
1Â protease and phosphatase inhibitor to the sample and mix
well (micropipette aspiration).
2. Homogenize the samples using 20 strokes of tissue homogenizer. Ensure that the sample remains on ice during the process.
3. Centrifuge at 500 Â g for 10 min at 4
C and collect the
supernatant (dissolved, solubilized protein). The extracted
protein lysate can be stored in À20
C prior use or in À80
C
for long-term storage.
4. Perform protein quantitation using BCA assay with the
Thermo Scientific Pierce BCA Protein Assay Kit (see Note 5).
3.2 Protein
Identification:
in-Solution Trypsin
Digestion and Tandem
Mass Spectrometry
LC-MS/MS
3.2.1 In-solution Trypsin
Digestion
1. Reconstitute protein samples in ddH 2 O. Aliquot approximately 100 μg of proteins (estimated from the protein assay
kit) in 100 μL from each sample and add into a 1.5 mL protein
lo-bind centrifuge tube.
2. Mix 25 μL of 100 mM ABC, 25 μL of TFE, and 1.0 μL of DTT
to the samples and vortex briefly (10 s). Heat the mixture for
20 min at 90
C and cool down to room temperature (see Note
6).
3. Add 4 μL of IAM (alkylation step) to the sample and incubate
at room temperature in the dark (avoid direct lighting/sunlight) for 1 h.
4. Add 1 μL of DTT to quench excess IAM and incubate at room
temperature in the dark for 1 h.
5. Add 300 μL of ddH 2 O and 100 μL of ABC to dilute the sample
and add 1 μL of trypsin. Incubate the sample overnight at
37
C.
6. Centrifuge the sample briefly (10 s), add 1 μL of formic acid to
stop the enzymatic reaction, and concentrate the sample using
a vacuum concentrator.
3.2.2 Peptide Clean-up/
Desalting
1. Desalt and reconcentrate the digested peptides using Pierce
C18 Spin Column (ThermoFisher, USA).
2. Prepare sample (digested proteins) by mixing 3 parts sample to
1 part sample buffer.
3. Tap the spin column gently to settle resin, remove the top and
bottom cap of the spin column, and place the column into a
receiver tube (1.5 mL microcentrifuge tube).
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Syafiq Asnawi Zainal Abidin et al.
3. Buffer B: 0.1% FA in 90% ACN.
3 Methods
3.1 Protein
Extraction
and Quantification
1. Add approximately 200 μL of RIPA buffer (see Note 4) with
1Â protease and phosphatase inhibitor to the sample and mix
well (micropipette aspiration).
2. Homogenize the samples using 20 strokes of tissue homogenizer. Ensure that the sample remains on ice during the process.
3. Centrifuge at 500 Â g for 10 min at 4
C and collect the
supernatant (dissolved, solubilized protein). The extracted
protein lysate can be stored in À20
C prior use or in À80
C
for long-term storage.
4. Perform protein quantitation using BCA assay with the
Thermo Scientific Pierce BCA Protein Assay Kit (see Note 5).
3.2 Protein
Identification:
in-Solution Trypsin
Digestion and Tandem
Mass Spectrometry
LC-MS/MS
3.2.1 In-solution Trypsin
Digestion
1. Reconstitute protein samples in ddH 2 O. Aliquot approximately 100 μg of proteins (estimated from the protein assay
kit) in 100 μL from each sample and add into a 1.5 mL protein
lo-bind centrifuge tube.
2. Mix 25 μL of 100 mM ABC, 25 μL of TFE, and 1.0 μL of DTT
to the samples and vortex briefly (10 s). Heat the mixture for
20 min at 90
C and cool down to room temperature (see Note
6).
3. Add 4 μL of IAM (alkylation step) to the sample and incubate
at room temperature in the dark (avoid direct lighting/sunlight) for 1 h.
4. Add 1 μL of DTT to quench excess IAM and incubate at room
temperature in the dark for 1 h.
5. Add 300 μL of ddH 2 O and 100 μL of ABC to dilute the sample
and add 1 μL of trypsin. Incubate the sample overnight at
37
C.
6. Centrifuge the sample briefly (10 s), add 1 μL of formic acid to
stop the enzymatic reaction, and concentrate the sample using
a vacuum concentrator.
3.2.2 Peptide Clean-up/
Desalting
1. Desalt and reconcentrate the digested peptides using Pierce
C18 Spin Column (ThermoFisher, USA).
2. Prepare sample (digested proteins) by mixing 3 parts sample to
1 part sample buffer.
3. Tap the spin column gently to settle resin, remove the top and
bottom cap of the spin column, and place the column into a
receiver tube (1.5 mL microcentrifuge tube).
236
Syafiq Asnawi Zainal Abidin et al.