The Cofactor Analysis workflow

The Cofactor Analysis workflow is designed to identify proteoforms from top-down mass spectrometry and is identical to the Subunits analysis for the Complex-Down workflow.

The Cofactor Analysis workflow is partitioned into the Proteoform Analysis and Composition tabs.

Create the Proteoform Analysis

Procedure

  1. From the Home screen, select the Cofactor Analysis workflow.
  2. Select the Proteoform Analysis tab from the left panel to start the analysis.
  3. Load a data file and select spectra scans.
  4. Deconvolute the data as follows:
    1. Select a region on the Chromatogram to begin automatic deconvolution.
      The resulting masses display as a list under Subunit Masses.
    2. To adjust the deconvolution settings, select Deconvolution Settings. The adjusted deconvolution will automatically overwrite existing Subunit Masses. To learn more, see Deconvolution algorithms.
    3. To add a custom mass, select Add Mass.
    4. Adjust the Search Settings if needed.
  5. Select subunit masses for the proteoform search as follows:
    1. Select individual masses under Subunit Masses by selecting the plus sign next to a mass.
      The selected masses are added to the Subunit Proteoforms search list.
    2. To overwrite existing selections in the Subunits Proteoforms search list, disable the Add to Search toggle. This feature is automatically enabled.
    3. To add all masses listed under Subunit Masses, select Add All to Search.
      All masses are added to the Subunit Proteoforms search list.
  6. Add fragmentation data as follows:
    1. Under Subunit Proteoforms, add associated fragmentation data by selecting the plus sign next to a given mass under the Fragmentation column. This allows the subunits to be identified using a database search.
      The Add Fragmentation Data dialog box displays.
    2. Under Data File, select Browse and upload a fragmentation file for the selected subunit.
    3. Specify information about the target that was fragmented, the fragmentation type, and restrict which groups to analyze using the Scan Filter dropdown.
      The following example specifies only scans with 300 eV HCD fragmentation to the 6184 M/Z target is displayed.
    4. Select an override fragmentation method determined by auto-search from the Fragmentation Method dropdown.
    5. Enter the Scan Range to specify the scans being averaged together. You can indicate a range with a dash (-) or enter a series of numbers with commas.
    6. Enter the Calibration to apply a shift to all spectral data.
    7. Select OK to return to the proteoform search.

Create the Composition analysis

Procedure

  1. Select the Composition tab from the left panel to start the analysis.
  2. From the Proteoforms table, select one of the masses.
  3. Adjust the mass tolerance in the Stoichiometry tab.
  4. Select the Edit button.
  5. Adjust the covalent masses and select the desired chemical entities to be active, then select Close.
  6. Select the Calculate button.
    The proteoform overview for the selected proteoform displays.
  7. Review the results in the Proteoform Composition.