Christie Hunter

Member

How are peptides and transitions selected for use in quantification, when importing from a ProteinPilot software group file – using SWATH acquisition microapp or Extractor in OneOmics suite?

Similar selection processes are used in both the SWATH acquisition microapp in PeakView software on the desktop and the Extractor app in OneOmics suite.

During the protein grouping process in ProteinPilot software, there is an extensive analysis that is done on the proteins and peptides that are identified. These results are annotated according to what peptides are shared between proteins and which spectrum best represent the peptide.  This information can be extracted during library extraction, along with other information that is used for peptide and fragment selection. When starting data processing, the user defines a range of filter criteria including peptide confidence, exclusion of shared or modified peptides, and how many peptides per protein and fragments per peptide. From the list of peptides from the group file that have not been excluded based on these filters, the peptides for extraction are selected from highest to lowest MS1 intensity.

In terms of selecting which fragment ions to use for each peptide, we assign 3 tiers of fragments for selection. The first tier is the fragment ions above the Q1 selection window, and ions are chosen in from high to low intensity. If more ions are needed based on the user defined # of fragments per peptide, the next tier of ions that are selected from are the ion below the Q1 selection window and above the y3/b3 ion, again from high to low intensity.  That last tier of fragment ions is taken from within the Q1 selection window. Currently only y and b ions are used for extraction.

RUO-MKT-18-13709-A

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