3.5 In-Gel Protein
Digestion
1. Protein bands are excised manually and transferred to 2.5-mL
LoBind tubes.
2. The gel pieces are washed twice with Milli-Q water and with
50% (v/v) ACN/25 mM Ambic until the blue color disappears.
3. Then, the gels are dehydrated with ACN.
4. Gel pieces are reduced with 10 mM DTT in 50 mM Ambic for
30 min at 56
C.
5. Repeat steps 2 and 3.
6. Gel pieces are alkylated with 55 mM IAA in 50 mM Ambic for
20 min in darkness.
7. Repeat steps 2 and 3.
8. 30 μL of trypsin (20 ng/μL in 12.5 mM Ambic/2% (v/v)
ACN) is added to the gel and incubated for 60 min at 0
C.
9. The bands are covered with 100 μL of 12.5 mM Ambic and
incubated for 12 h at 37
C.
10. The next day, 50 μL of 5% (v/v) formic acid is added, and the
supernatant is transferred to a new Lo-Bind tube.
11. The peptides are further extracted from the gel twice with 50%
(v/v) ACN/0.1% (v/v) TFA (Â3) and ACN (Â1).
12. The samples are dried-down and stored at À20
C until
their use.
3.6 SWATH-MS
Methods
3.6.1 LC-MS Data
Acquisition
1. Online reversed-phase (RP) LC separation prior to MS analysis
is performed using the Nano-LC Ultra system Eksigent
®
425 in micro mode, coupled with the ekspert™
400 autosampler.
2. From each pool of samples, 4 μg of digested peptides is injected
in a precolumn using a flow rate of 10 μL/min concentrated
and desalted for 3 min in a 200 Â 0.5 mm precolumn-YMC,
3 μm 120 A ˚ .
3. Subsequently, the reverse-phase separation is performed on the
0.5 Â 150 cm analytical column, 3 μm 120 A ˚ at a flow rate of
5 μL/min. The gradient is developed during 30 min from 5% A
to 30% B (A: is Milli-Q water plus 0.1% formic acid and B is
ACN plus 0.1% formic acid) (see Notes 1 and 7). The column is
coupled to the micro ESI source using a 50-μm ID probe. The
eluted peptides are analyzed on a High-Resolution Quadrupole Time-of-Flight (QTOF instrument), TripleTOF
® 6600+.
Instrument Quality Control
Considerations [25, 119]
Standard operating procedures are run prior to sample analysis in
order to monitor instrument performance and achieve the maximum reproducibility and reliability of the analysis. This routine
encompasses the run of different standards to assess the efficiency
of both chromatography and mass spectrometry (in our case, we
use 4 μg of a tryptic digest of a K562 extract (SciexSWATH-MS
128
Maria del Pilar Chantada-Va ´ zquez et al.
Digestion
1. Protein bands are excised manually and transferred to 2.5-mL
LoBind tubes.
2. The gel pieces are washed twice with Milli-Q water and with
50% (v/v) ACN/25 mM Ambic until the blue color disappears.
3. Then, the gels are dehydrated with ACN.
4. Gel pieces are reduced with 10 mM DTT in 50 mM Ambic for
30 min at 56
C.
5. Repeat steps 2 and 3.
6. Gel pieces are alkylated with 55 mM IAA in 50 mM Ambic for
20 min in darkness.
7. Repeat steps 2 and 3.
8. 30 μL of trypsin (20 ng/μL in 12.5 mM Ambic/2% (v/v)
ACN) is added to the gel and incubated for 60 min at 0
C.
9. The bands are covered with 100 μL of 12.5 mM Ambic and
incubated for 12 h at 37
C.
10. The next day, 50 μL of 5% (v/v) formic acid is added, and the
supernatant is transferred to a new Lo-Bind tube.
11. The peptides are further extracted from the gel twice with 50%
(v/v) ACN/0.1% (v/v) TFA (Â3) and ACN (Â1).
12. The samples are dried-down and stored at À20
C until
their use.
3.6 SWATH-MS
Methods
3.6.1 LC-MS Data
Acquisition
1. Online reversed-phase (RP) LC separation prior to MS analysis
is performed using the Nano-LC Ultra system Eksigent
®
425 in micro mode, coupled with the ekspert™
400 autosampler.
2. From each pool of samples, 4 μg of digested peptides is injected
in a precolumn using a flow rate of 10 μL/min concentrated
and desalted for 3 min in a 200 Â 0.5 mm precolumn-YMC,
3 μm 120 A ˚ .
3. Subsequently, the reverse-phase separation is performed on the
0.5 Â 150 cm analytical column, 3 μm 120 A ˚ at a flow rate of
5 μL/min. The gradient is developed during 30 min from 5% A
to 30% B (A: is Milli-Q water plus 0.1% formic acid and B is
ACN plus 0.1% formic acid) (see Notes 1 and 7). The column is
coupled to the micro ESI source using a 50-μm ID probe. The
eluted peptides are analyzed on a High-Resolution Quadrupole Time-of-Flight (QTOF instrument), TripleTOF
® 6600+.
Instrument Quality Control
Considerations [25, 119]
Standard operating procedures are run prior to sample analysis in
order to monitor instrument performance and achieve the maximum reproducibility and reliability of the analysis. This routine
encompasses the run of different standards to assess the efficiency
of both chromatography and mass spectrometry (in our case, we
use 4 μg of a tryptic digest of a K562 extract (SciexSWATH-MS
128
Maria del Pilar Chantada-Va ´ zquez et al.