The Complex-Down workflow

The Complex-Down workflow is designed for determining the composition of a protein complex with mass deconvolution, proteoform search, and stoichiometry calculation.

The Complex-Down workflow is partitioned into the Complex, Subunits, and Composition tabs.

Create the Complex analysis

Procedure

  1. From the Home screen, select the Complex-Down workflow.
  2. Select the Complex tab from the left panel to start your 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 Complex Masses.
    2. To view and average a selection of spectra from the Chromatogram, right-click anywhere on the Chromatogram display and drag to select the range of interest.
      The scans with the same MS level in the selected region are averaged together and displayed in the Spectrum viewer (bottom, center panel), which can toggle between the M/Z Spectrum and Mass Spectrum views.
    3. To change the display or specify selections for deconvolution, select Options in the Spectrum viewer.
    4. To zoom, select anywhere on the chromatogram and drag the mouse.
    5. To view charge states for a given mass, select View Charge States.
    6. To add a custom mass, select Add Mass.
    7. To Change the MSn level or define the scan range for the chromatogram, select Options in the Chromatogram viewer.
  5. If no mass results show, then adjust the deconvolution settings. For example, the mass range might be too low. See Adjust the deconvolution settings for more information.

Create the Subunits analysis

The Chromatogram and Spectrum in the Subunits tab behave similarly to the Complex tab.

Procedure

  1. Select the Subunits tab from the left panel to continue your complex-down analysis.
  2. Load a data file and select spectra scans.
  3. Deconvolute the data as follows:
    1. To begin automatic deconvolution, select a region on the Chromatogram.
      The resulting masses display as a list under Subunit Masses.
    2. To adjust the deconvolution, select Deconvolution Settings.
      The adjusted deconvolution automatically overwrites existing Subunit Masses. To learn more, see Deconvolution algorithms.
    3. To add a custom mass, select Add Mass.
  4. Select the 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.
  5. 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.
  6. Search for proteoforms as follows:
    1. Select the Select from Available Databases button to enter a database to search against.
    2. In the Add Proteoforms from Databases dialog box, select an existing database or create a new one, then select the Select button.
    3. Select Search under Subunit Proteoforms to start the proteoform search.
  7. To adjust the search settings, select Search Settings and specify the necessary parameters. For more information, see Adjust the Search Settings.
    The resulting fragment maps for proteoforms display below the search.
  8. Analyze the proteoform results. Select the corresponding links to access the following additional analytical tools:

    • TDValidator: Perform robust targeted analysis of a single proteoform or multiple proteoforms.

    • ProSight Lite (PS Lite): Match a single candidate sequence and its modifications against a set of mass spectrometric observations and product publication-ready fragment maps.

    • Structure Viewer (Structure): Superimpose top-down fragment results onto three-dimensional proteoform structures.

Create the Composition analysis

Procedure

  1. Select the Composition tab from the left panel to set up stoichiometry calculations for your complex.
    The right panel populates with the complexes and associated subunit masses from the previous tabs, under their respective sections.
  2. Specify the complex and subunits as follows:
    1. Select a mass from the Complexes list.
    2. Under Subunits, specify the number of subunits to allow for each complex.
      ProSight Native automatically caps the number of subunits so that the total mass does not exceed the complex mass.
    3. To apply an allowable range to all subunits, enter a Global Subunit Range and select Refresh.
    4. To lock the range for the given subunit, select Lock.
    5. To include non-covalent masses in the stoichiometry calculation, enable the Include Non-Covalent Masses toggle.
    6. To edit, select Edit.
      A new dialog box displays with options for including masses from the Chemical Entities of Biological Interest or UniProt.
  3. Calculate the stoichiometry as follows:
    1. Enter the Mass Tolerance for All Components.
    2. Select Calculate to determine the possible stoichiometries of the complex.
    3. To include known interactors with the complex from UniProt in your calculation, enable the Include Known Interactors from UniProt toggle.
      The resulting stoichiometries display as a list under Stoichiometry.
    4. To view a composition for the complex, select any result under Stoichiometry.
      The composition displays under Composition.