3.6 MS Acquisition
in SWATH Mode
1. Sample loading in the Evotips:
(a) Activate C18 material of Evotips. Place Evotips in the
Evosep rack holder on top of a 96 well plate containing
in each well 100 μL of 2-propanol. Soak the tips for a
minimum of 10 s.
(b) Add 20 μL of MS buffer B to all Evotips and centrifuge at
700 Â g for 1 min.
(c) Soak Evotips with 2-propanol and add 20 μL of MS
buffer A.
(d) Place Evotips within the yellow rack in their original box
(see Note 16). Add 10 μL of MS buffer A to all Evotips
and shortly spin for 10 s. Load SEC-fractions and centrifuge at 700 Â g for 1 min.
(e) For washing, add 100 μL of MS buffer A on top of the
Evotips and centrifuge at 700 Â g for 3 min.
(f) The samples are ready for injection. To avoid drying up of
the C18 material, add 200 μL of MS buffer A on top of the
tips (see Note 17) and briefly spin at 200 Â g for 10 s to
remove any air bubble that might be present.
2. Interfacing Evosep One to TripleTOF 6600:
(a) Interface the PepSep C-18 LC column to the
MS-TripleTOF 6600 by connecting the column to the
emitter through the metal union (Union Assembly ZDV
Valco Type).
(b) Create in the Analyst main panel “Hardware Configuration Editor” one new “Hardware Profile” including only
the TripleTOF 6600, and remove other hardware devices
(e.g., syringe pumps, any LC pumps) from the settings, to
allow contact closure triggering via Chronos.
(c) Start Chronos, and select the method “Prepare” and run
methods “Degas,” “Purge” and equilibrate the column by
running the “Align solvents” (see Note 18). After these
preparation steps, the system is ready for measurements.
3. Evosep gradient.
The Evosep One system comes with five prefixed methods
and proposed commercial columns for each method (see
Table 3 for a summary). For the analysis of SEC-Fractions in
SWATH mode, the “60 samples per day” method is the best
trade-off between throughput and number of proteins identified, saving 75% of the acquisition time, while retaining the
majority of the proteins identified compared to a standard
60 min nanoLC separation using default settings on a Triple
TOF 6600 [23].
System-Wide Profiling of Protein Complexes Via Size Exclusion. . .
281
in SWATH Mode
1. Sample loading in the Evotips:
(a) Activate C18 material of Evotips. Place Evotips in the
Evosep rack holder on top of a 96 well plate containing
in each well 100 μL of 2-propanol. Soak the tips for a
minimum of 10 s.
(b) Add 20 μL of MS buffer B to all Evotips and centrifuge at
700 Â g for 1 min.
(c) Soak Evotips with 2-propanol and add 20 μL of MS
buffer A.
(d) Place Evotips within the yellow rack in their original box
(see Note 16). Add 10 μL of MS buffer A to all Evotips
and shortly spin for 10 s. Load SEC-fractions and centrifuge at 700 Â g for 1 min.
(e) For washing, add 100 μL of MS buffer A on top of the
Evotips and centrifuge at 700 Â g for 3 min.
(f) The samples are ready for injection. To avoid drying up of
the C18 material, add 200 μL of MS buffer A on top of the
tips (see Note 17) and briefly spin at 200 Â g for 10 s to
remove any air bubble that might be present.
2. Interfacing Evosep One to TripleTOF 6600:
(a) Interface the PepSep C-18 LC column to the
MS-TripleTOF 6600 by connecting the column to the
emitter through the metal union (Union Assembly ZDV
Valco Type).
(b) Create in the Analyst main panel “Hardware Configuration Editor” one new “Hardware Profile” including only
the TripleTOF 6600, and remove other hardware devices
(e.g., syringe pumps, any LC pumps) from the settings, to
allow contact closure triggering via Chronos.
(c) Start Chronos, and select the method “Prepare” and run
methods “Degas,” “Purge” and equilibrate the column by
running the “Align solvents” (see Note 18). After these
preparation steps, the system is ready for measurements.
3. Evosep gradient.
The Evosep One system comes with five prefixed methods
and proposed commercial columns for each method (see
Table 3 for a summary). For the analysis of SEC-Fractions in
SWATH mode, the “60 samples per day” method is the best
trade-off between throughput and number of proteins identified, saving 75% of the acquisition time, while retaining the
majority of the proteins identified compared to a standard
60 min nanoLC separation using default settings on a Triple
TOF 6600 [23].
System-Wide Profiling of Protein Complexes Via Size Exclusion. . .
281