The following figure shows an overview of the CHIMERYS workflow.

The CHIMERYS workflow contains four main steps, where the workflow does the following:

  1. Performs a first pass search.
  2. Recalibrates the masses, the retention times, and the fragmentation energies based on results of the first pass search.
  3. Performs a first main search, which deconvolutes each spectrum and produces a list of peptide spectrum matches (PSMs) for each MS/MS spectrum.
  4. Calculates the FDR for all PSMs using Mokapot1).

These four main steps are performed for DDA, DIA, and PRM datasets. However, for DIA and PRM data, CHIMERYS performs the following extra steps:

  1. Combines PSMs regardless of their confidence with any PSMs from the CHIMERYS Inclusion File (if one was selected).
  2. Performs a second main search with the search space restricted to the combined list of PSMs.
  3. For more information on how to create an inclusion file, see Export CHIMERYS Inclusion File.
  4. Calculates FDR for all PSMs using Mokapot.
  5. Calculates peak areas based on the CHIMERYS coefficients for identified peptide precursors in a raw file-specific integration window around their elution apexes. Elution apexes and the integration window are determined based on FDR-controlled PSMs.
  6. Only one of the PSMs for a peptide needs to be of high confidence, while lower confidence identifications in adjacent scans will be used to extend peptide elution profiles. CHIMERYS will return a trapezoidal approximation of the area beneath the elution profile as shown in the following figure.

NOTE

All peaks in the chromatogram require for their quantification that the peptide is identified as a PSM in that scan. CHIMERYS is thus a spectrum-centric, untargeted method for MS2-based quantification.

NOTE

If you select the One Job For All Files parameter in the CHIMERYS node, CHIMERYS will also output peak areas for peptide precursors of medium or low confidence as long as they were identified with high confidence in at least one of the other files searched together in the Processing Workflow. Conceptually, this is similar to what is commonly referred to as Match Between Runs (or MBR by other software).

The results are then sent back to Proteome Discoverer from the CHIMERYS node.