3.9 FASP Digestion
1. Before digestion, prepare the filters of the Amicon™ Ultra-0.5.
Wash the membranes with 400μL of 0.1 M NaOH at
14,000 Â g for 10 min. Add another wash with 400μL ultrapure water and centrifugate it.
2. Prepare the samples: thaw them for 10 min using Eppendorf
Thermomixer
® comfort at 600 rpm and 20
C.
3. Add 0.1 M DTT in order to obtain 20 mM (final concentration). Heat samples for 5 min at 99
C in the Thermomixer.
4. Temper the samples before adding UA buffer. Dilute samples
with UA buffer to obtain less than 0.1% SDS (final concentration) (see Notes 10 and 11).
5. Add the samples into the filter and then centrifuge for 15 min
at 14,000 Â g. The maximum volume inside the filter is 450μL.
Do as many steps as needed in order to introduce all samples
into the filter.
6. Wash the filter two times with 200μL UA buffer and centrifuge
them for 15 min at 14,000 Â g.
7. Alkylation step: add 0.05 M IAA for 20 min with gentle agitation (400 rpm).
8. Wash two times the filter with 200μL UA buffer and then four
times with TEAB buffer. All steps are followed with a centrifugation for 15 min at 14,000 Â g (see Note 12).
9. Digest with trypsin (5:100) (v/v) for 18 h with gentle agitation
at 37
C in the dark.
10. The second day, elute the samples with 100μL TEAB (threetimes) and centrifuge for 15 min at 14,000 Â g.
11. Evaporate the elute with a SpeedVac and resuspend the
digested peptides in 20μL of 5% methanol; 0.5% TFA.
3.10 LC-MS/MS
Analysis
In this example, samples are analyzed using LC coupled to an LTQ
XL Orbitrap equipped with a nanoESI ion source.
1. Dilute the sample to a final volume of 20μL and load it into the
chromatographic system consisting of a C18 preconcentration
connected to a 15-cm-long, 100-μm-i.d. C18 column.
2. Separate them using 0.4μL/min in a 120-min acetonitrile
gradient from 3% to 35% of solvent A using an Agilent 1200
nanoflow system (see Note 13).
3. The LC is coupled to an LTQ-Orbitrap XL mass spectrometer
equipped with a nanoelectrospray ion source (see Note 14).
4. Use the database search Proteome Discoverer v1.4 and SwissProt human database in order to identify proteins.
20
Maria N. Barrachina and A ´ ngel Garcı ´a
1. Before digestion, prepare the filters of the Amicon™ Ultra-0.5.
Wash the membranes with 400μL of 0.1 M NaOH at
14,000 Â g for 10 min. Add another wash with 400μL ultrapure water and centrifugate it.
2. Prepare the samples: thaw them for 10 min using Eppendorf
Thermomixer
® comfort at 600 rpm and 20
C.
3. Add 0.1 M DTT in order to obtain 20 mM (final concentration). Heat samples for 5 min at 99
C in the Thermomixer.
4. Temper the samples before adding UA buffer. Dilute samples
with UA buffer to obtain less than 0.1% SDS (final concentration) (see Notes 10 and 11).
5. Add the samples into the filter and then centrifuge for 15 min
at 14,000 Â g. The maximum volume inside the filter is 450μL.
Do as many steps as needed in order to introduce all samples
into the filter.
6. Wash the filter two times with 200μL UA buffer and centrifuge
them for 15 min at 14,000 Â g.
7. Alkylation step: add 0.05 M IAA for 20 min with gentle agitation (400 rpm).
8. Wash two times the filter with 200μL UA buffer and then four
times with TEAB buffer. All steps are followed with a centrifugation for 15 min at 14,000 Â g (see Note 12).
9. Digest with trypsin (5:100) (v/v) for 18 h with gentle agitation
at 37
C in the dark.
10. The second day, elute the samples with 100μL TEAB (threetimes) and centrifuge for 15 min at 14,000 Â g.
11. Evaporate the elute with a SpeedVac and resuspend the
digested peptides in 20μL of 5% methanol; 0.5% TFA.
3.10 LC-MS/MS
Analysis
In this example, samples are analyzed using LC coupled to an LTQ
XL Orbitrap equipped with a nanoESI ion source.
1. Dilute the sample to a final volume of 20μL and load it into the
chromatographic system consisting of a C18 preconcentration
connected to a 15-cm-long, 100-μm-i.d. C18 column.
2. Separate them using 0.4μL/min in a 120-min acetonitrile
gradient from 3% to 35% of solvent A using an Agilent 1200
nanoflow system (see Note 13).
3. The LC is coupled to an LTQ-Orbitrap XL mass spectrometer
equipped with a nanoelectrospray ion source (see Note 14).
4. Use the database search Proteome Discoverer v1.4 and SwissProt human database in order to identify proteins.
20
Maria N. Barrachina and A ´ ngel Garcı ´a