17. It is possible to store loaded samples for a few days
($1–2 weeks) in the Evotips, without affecting sample quality.
Keep them at 4
C with a layer of MS buffer A on top. The thin
layer of MS buffer A that remains on top the C18 resin ensures
that the tips remain wet during the sample loading step.
18. Running the “Prepare” method is necessary every time the
instrument has been idle (no column flow). First run the
“Degas” cycle to remove any air and then schedule “Prepare”
without any option ticked on to check column pressure, possible leaking, and flush the column.
19. For the calibration method, use the following TripleTOF 6600
settings:
(a) Mass spectrometer period duration: 11.502 min.
(b) TOF masses (Da) (precursors ions range): 350–1500 m/
z.
(c) Cycle time: 0.5499 s.
(d) TOF accumulation time: 0.250 s.
(e) Product Ion (729.3) scan range: 350–1500 m/z.
(f) Product Ion (729.3) accumulation time: 0.250 s.
(g) High sensitivity scan mode.
(h) Parameter settings:
l
Declustering potential (DP): 100.
l
Collision energy (CE): 45.0.
l
Collision energy spread (CES): 5.0 eV.
l
Ion release delay (IRD): 30.
l
Ion release width (IRW): 15.
20. We recommend performing targeted data extraction with
OpenSWATH or with other tools such as Spectronaut [36]
and DIAumpire [37]. OpenSWATH uses for extraction of
quantitative data from SWATH measurements, the spectral
assay library with corresponding RT and performs retention
time normalization with the iRT-peptides or a set of endogenous RT peptides. Evosep One SWATH data are characterized
by nonlinearly distributed RT of iRT peptides across the run.
Thus, it is necessary to adapt the retention time for all assays in
order to use the retention time normalization applied for the
OpenSWATH workflow. For this, we use a loess (locally estimated scatterplot smoothing) model, and predict the retention
times for all assays in a spectral assay library. The adapted library
can then be converted to *pqp library applying the OpenMS
tool “TargetedFileConverter.”
21. It is highly recommended to generate a sample-specific spectral
library using high pH fractionated SEC input lysate samples,
following the published workflow for generating a high-quality
System-Wide Profiling of Protein Complexes Via Size Exclusion. . .
291
($1–2 weeks) in the Evotips, without affecting sample quality.
Keep them at 4
C with a layer of MS buffer A on top. The thin
layer of MS buffer A that remains on top the C18 resin ensures
that the tips remain wet during the sample loading step.
18. Running the “Prepare” method is necessary every time the
instrument has been idle (no column flow). First run the
“Degas” cycle to remove any air and then schedule “Prepare”
without any option ticked on to check column pressure, possible leaking, and flush the column.
19. For the calibration method, use the following TripleTOF 6600
settings:
(a) Mass spectrometer period duration: 11.502 min.
(b) TOF masses (Da) (precursors ions range): 350–1500 m/
z.
(c) Cycle time: 0.5499 s.
(d) TOF accumulation time: 0.250 s.
(e) Product Ion (729.3) scan range: 350–1500 m/z.
(f) Product Ion (729.3) accumulation time: 0.250 s.
(g) High sensitivity scan mode.
(h) Parameter settings:
l
Declustering potential (DP): 100.
l
Collision energy (CE): 45.0.
l
Collision energy spread (CES): 5.0 eV.
l
Ion release delay (IRD): 30.
l
Ion release width (IRW): 15.
20. We recommend performing targeted data extraction with
OpenSWATH or with other tools such as Spectronaut [36]
and DIAumpire [37]. OpenSWATH uses for extraction of
quantitative data from SWATH measurements, the spectral
assay library with corresponding RT and performs retention
time normalization with the iRT-peptides or a set of endogenous RT peptides. Evosep One SWATH data are characterized
by nonlinearly distributed RT of iRT peptides across the run.
Thus, it is necessary to adapt the retention time for all assays in
order to use the retention time normalization applied for the
OpenSWATH workflow. For this, we use a loess (locally estimated scatterplot smoothing) model, and predict the retention
times for all assays in a spectral assay library. The adapted library
can then be converted to *pqp library applying the OpenMS
tool “TargetedFileConverter.”
21. It is highly recommended to generate a sample-specific spectral
library using high pH fractionated SEC input lysate samples,
following the published workflow for generating a high-quality
System-Wide Profiling of Protein Complexes Via Size Exclusion. . .
291