assay library [38]. It is possible to use a community-based
repository assay library, such as the PanHuman library [39]
or a subset of a community-based library. For this example, the
PanHuman library was subset to proteotypic assays only, as the
search space for all assays within the PanHuman library would
be too large.
22. For SWATH data acquired on a TripleTOF 6600 interfaced
with the Evosep One system, some of the default parameters
need to be modified. The mz_extraction window is increased
to 100 ppm, as manual calibration is performed on a daily basis,
and thus the mass error might shift to a larger extent compared
to standard SWATH acquisitions. The rt_extraction_window is
set to 360, which extracts peaks around the library RT Æ180 s.
The OSW output files are in the SQLite format, which is still
experimental but lowers computation time for the subsequent
scoring steps. As the OSW is computationally and time
demanding, it might be useful to conduct the analysis on a
cluster or cloud infrastructure.
23. The commands for scoring of large-scale experiments are outlined in the http://www.openswath.org/en/latest/docs/
pyprophet.html#scaling-up “scaling-up” section.
24. The files are scored, and in addition, statistical reports for each
file/run on peptide level and for the experiment on protein
level in the form of a PDF file are generated. It is advised to
investigate this report to double-check the assay and decoy
distributions.
25. A moderate alignment_score threshold, which defines the minimal feature score needed to align between runs and a nonconservative fixed 0.05-m score cutoff for each single run
(FDR_cutoff) is used for Evosep-SWATH data. Peptide and
protein FDR across the SEC gradient is controlled at 1% with
the sibling peptide correlation filter function in the CCprofiler
package.
26. Timing:
1 Day: flushing of the column, measure a standard, flush in
SEC mobile phase overnight.
2 Day: preparation of primer sample, preparation of samples, and injection of primer sample and subsequently the
sample(s). 3 Day: FASP protocol and overnight digestion.
4 Day: fraction collection and drying of samples. 5 Day: loading of samples on Evotips and sample acquisition in DIA mode.
One week or less for sample analysis depending on the
amount of samples. The protocol can also be used with DDA
methods or other DIA analysis pipelines. Injection time with
longer gradients, however, substantially increase the
acquisition time.
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Andrea Fossati et al.
repository assay library, such as the PanHuman library [39]
or a subset of a community-based library. For this example, the
PanHuman library was subset to proteotypic assays only, as the
search space for all assays within the PanHuman library would
be too large.
22. For SWATH data acquired on a TripleTOF 6600 interfaced
with the Evosep One system, some of the default parameters
need to be modified. The mz_extraction window is increased
to 100 ppm, as manual calibration is performed on a daily basis,
and thus the mass error might shift to a larger extent compared
to standard SWATH acquisitions. The rt_extraction_window is
set to 360, which extracts peaks around the library RT Æ180 s.
The OSW output files are in the SQLite format, which is still
experimental but lowers computation time for the subsequent
scoring steps. As the OSW is computationally and time
demanding, it might be useful to conduct the analysis on a
cluster or cloud infrastructure.
23. The commands for scoring of large-scale experiments are outlined in the http://www.openswath.org/en/latest/docs/
pyprophet.html#scaling-up “scaling-up” section.
24. The files are scored, and in addition, statistical reports for each
file/run on peptide level and for the experiment on protein
level in the form of a PDF file are generated. It is advised to
investigate this report to double-check the assay and decoy
distributions.
25. A moderate alignment_score threshold, which defines the minimal feature score needed to align between runs and a nonconservative fixed 0.05-m score cutoff for each single run
(FDR_cutoff) is used for Evosep-SWATH data. Peptide and
protein FDR across the SEC gradient is controlled at 1% with
the sibling peptide correlation filter function in the CCprofiler
package.
26. Timing:
1 Day: flushing of the column, measure a standard, flush in
SEC mobile phase overnight.
2 Day: preparation of primer sample, preparation of samples, and injection of primer sample and subsequently the
sample(s). 3 Day: FASP protocol and overnight digestion.
4 Day: fraction collection and drying of samples. 5 Day: loading of samples on Evotips and sample acquisition in DIA mode.
One week or less for sample analysis depending on the
amount of samples. The protocol can also be used with DDA
methods or other DIA analysis pipelines. Injection time with
longer gradients, however, substantially increase the
acquisition time.
292
Andrea Fossati et al.