The Feature Finder is an algorithm design to scan the MS1 domain for all existent peptides in a sample. In a typical data-dependent acquisition assay, peptides are identified only if an MS2 scan is triggered from a precursor ion in an MS1 scan.
Therefore, if a peptide ion does not trigger an MS2 scan, or its score excludes it from identification, it will not be detected. Feature Finder ensures that a peak will not go undetected by identifying all possible isotopic distributions in a sample that could potentially be a peptide.
Feature Finder also allows the user to match these features to the current identifications and to add a specified number of the unidentified features as unknowns to the project. This process generates XICs for the unknowns and allow reporting on features such as retention time, mass, intensity, and fold change across samples.
Settings:
Users can enable Feature Finder and configure various settings, including minimum isotope correlation, mass range, maximum feature count, and minimum intensity, among others.
Feature Finder settings inside Peptides worklows
- Enable Feature Finder - The user can click to select between No and Yes to enable Feature Finder.
- Minimum Isotope Corr. - The user can set the minimum cutoff to match the found isotopic distribution to the theoretical distribution using Pearson's correlation. If a feature has an isotopic distribution that is vastly different from its theoretical, this can be used to exclude it.
- Mass Range Min - The user can set the minimum mass for a feature to be considered.
- Mass Range Max - The user can set the maximum mass for a feature to be considered.
- Maximum Features Count - The user can set the maximum feature count to include the most intense features.
- Absolute minimum intensity - The user can set the minimum intensity for a feature to be considered.
- Minimum Isotope Count - The user can set the minimum number of isotope peaks required for a feature to be considered. This applies only if its charge is greater than 2.
- Minimum Peak Width (min) - The user can set the minimum peak width in minutes for a feature to be considered.
- Minimum S/N Ratio - The user can set the minimum S/N ratio for a feature to be considered.
- Minimum Scan Count - The user can set the minimum scans required across the peak for a feature to be considered.
- Exclude +1 Charge Only Features - The user can click to select between Yes and No to exclude singly charged features.
- Exclude Features With MS2 Matches - The user can click to select between No and Yes to exclude features with MS2 matches. Exclude will remove the feature if it coincides within the time and mass tolerances set – it will not be included in the top X features to avoid duplicates. It is recommended to check this box for projects containing MS2 data and Byonic searches.
- Mass matching tolerance (ppm) and Time matching tolerance (min) - These parameters are used to match and remove features that have already been identified by MS2 (when Exclude Features With MS2 Matches is set to Yes) so that they do not contribute to the maximum feature count. They help to match the retention time and mass of an unknown feature to a MS2 feature so that it may be excluded from the Peptides table in Peptide Analysis.
Feature Finder output results: the Unknown impurities will be tracked and found as the screenshot above where there are description of m/z s RTs and you can appreciate Isotope and extracted ion chromatogram (XIC). NOTE: Unknow does no have MS2 information populated
REPORTING:
From the Report level, the Unknows could be compared from samples to samples. As you know PMI has a
lot of report capabilities, so you do not need to use Excel spreadsheet for doing comparison
Feature Finder report template (.rptc file) can be found under Presets / Report presets
In order to have comparison you got to have 1 reference vs Non Reference samples and the report will
automatically calculate fold change between them (see Max Fold Change)
Feature Finder report tab with Reference, Non-reference sample and Max fold change columns
NOTE: users can filtered out Fold change within report
Characterize Unknown using Peptide Manager:
Users can characterize those Unknows to Sequence using the Peptide Manager / Intercept function.
Reminder: Peptide Manager combines all of the In-silico functionalities from Edit > In-silico peptides with a color-coded preview of the project changes and enhanced control over which rows and columns are affected
Peptide Manager window
Procedure
Click Intersect CSV and select a CSV file:
Select a CSV file using the Browse button.
Select between the Matching options “Use mass and time” and “Use sequence and modifications”
Click OK to return to Peptide Manager and select columns to review. New values will be show in red.
Intersect CSV Peptide Manager using Mass and Time for Unkwown characterization
NOTE: you can export the Unknowns list to a csv file for another set of MS2 search using Inclusion List (Byonic)
New values will be show in red.
The below image displays expanded rows to showcase additional content
Unknown to recognized sequences using Intersect CSV Peptide Manager
Byonic MS2 seachInclusion List
The Inclusion tab allows the user to define a m/z ratio range and/or elution time range segments to include in the search. Import CSV file
Byonic Inclusion List for Unknow search
for Stand alone Byonic: Enter the begin and end values for one or both range types. A new row is automatically added for additional range definitions