The Spectrum Grouper node applies a grouping function to the data set. You can use this node to group spectra from different MS order scans or different mass analyzers.

The Spectrum Grouper node combines the MS/MS peak lists that belong to the same precursor species within specified tolerances. This feature can lead to a reduction in the number of peak lists, faster search times, and a higher quality of the individual peak lists.

The Spectrum Grouper steps through the peak lists that it receives and combines them according to defined criteria. The result of this grouping is a combined fragment spectrum peak list that is associated with a single precursor mass and charge. The peak lists are then used as the input for the peptide identification search.

When you define the grouping tolerances, keep in mind:

  • Setting the tolerances too wide leads to combining fragment spectra of multiple or unique species. This type of setting decreases the quality of the search results and can result in fewer peptide hits, more false positives, or overall lower probability scores.
  • Setting the grouping tolerances too tight can result in longer search times and a possible decrease in the quality of data, because species that could be combined are treated as unique.

In some cases, it might be beneficial to group spectra if a low-abundance peptide produces a poor-quality (low signal-to-noise) spectrum.

You can also use the Spectrum Grouper node to group HCD and CID scans to improve the overall fragment ion coverage in alternating HCD/CID experiments. For this latter purpose, set the Allow MS Order Mismatch parameter to True. The default is False.

The Spectrum Grouper node cannot group spectra from multiple input files.

The following table describes the parameters for the Spectrum Grouper node.

Spectrum Grouper node parameters

Parameter

Description

Precursor Mass Criterion

Groups spectra measured within the given mass and retention-time tolerances into a single spectrum for analysis on the basis of a predesignated precursor mass criterion. Select one of the following:

  • (Default) Same Measured Mass-to-Charge: Averages the tandem mass spectrum of precursors that have the same mass-to-charge value within the specified tolerance.
  • Same Singly Charged Mass: Groups different charge states of the same precursor together. It averages the tandem mass spectrum of precursors that have the same singly charged mass within a specified tolerance. Applicable only when the charge state of the precursor is recorded in the original data, as would be the case with higher-resolution data such as FT ICR or Orbitrap data. The charge associated with the combined peak list is the highest charge of the precursors in the group.

Precursor Mass Tolerance

Specifies the mass window for which precursor ions are considered to be the same species and are therefore added to the same group. They must also elute within the specified mass tolerance window.

Range 0.0–3.5 Da or 0.0–5000 ppm; default: 10 ppm

Max. RT Difference [min]

Specifies the chromatographic window where precursors to be grouped must reside to be considered the same species and therefore added to the same group. For example, a precursor ion with a mass-to-charge ratio of 619 that elutes at 37.76 minutes is different from a precursor ion with a mass-to-charge ratio of 619 that elutes at 47.10 minutes.

Range: 0.0–no maximum; default: 1.5 minutes

Allow Mass Analyzer Mismatch

Determines whether the fragment spectrum for scans with the same precursor mass is grouped, regardless of mass analyzer and activation type.

  • True: Sorts the fragment spectrum for scans with the same precursor mass into the same group, regardless of mass analyzer and activation type. For example, both ITMS and FTMS MS/MS scans are sorted into the same group, as long as they have the same precursor mass.
  • (Default) False: Does not group the fragment spectrum for scans with the same precursor mass if they arise from different mass analyzers. These scans are analyzed separately.

Note When you attempt to group ITMS and FTMS scans, also use the Spectrum Normalizer node to average these two types of data since they are of very different intensities. The Spectrum Normalizer node normalizes for effective averaging. For information about the Spectrum Normalizer node, see Spectrum Normalizer node.

Allow MS Order Mismatch

Determines whether spectra from different MS order scans (for example, MS2 and MS3) can be grouped together. This option might be useful, for example, for analyzing MS2 + MS3 data from a data-dependent neutral loss experiment.

  • True: Groups spectra from different MS order scans together.
  • (Default) False: Does not group spectra from different MS order scans together.