The XlinkX/PD Detect node for cleavable crosslinkers detects spectra containing potential crosslinks on the basis of the characteristic peak doublets, with a constant delta mass resulting from the two fragmentation sites in the crosslink. The following table describes the parameters for this node.

  • Set the Acquisition Strategy parameter by selecting the acquisition mode used to acquire the raw data file.
  • For the Crosslink Modification parameter, select the appropriate crosslink modification.

If no modification is available, define the crosslink modification in the Chemical Modifications view:

  • Click the Crosslink tab in the Edit Chemical Modification dialog box to define the settings for the crosslink modification.
XlinkX/PD Detect node parameters

Parameter

Description

Acquisition Strategy

Specifies the acquisition mode used to acquire the data in the raw data file from the following options:

  • MS2: Specifies the MS2 only for the MS-cleavable mode of acquisition. The mass spectrometer fragments the precursor ions in a single approach.
  • MS2_MS2: Specifies the MS2_MS2 mode of acquisition, which is used only for cleavable crosslinkers. The mass spectrometer fragments every precursor ion twice, usually by CID with a lower energy. This fragmentation cleaves the crosslinker. The second fragmentation typically uses ETD (EThcD) to fragment the peptide backbone, where the crosslinker remains stable.
  • MS2_MS2_MS3: Specifies the MS2_MS2_MS3 mode of acquisition, which is used only for cleavable crosslinkers. This mode uses MS2 fragmentation spectra and MS3 fragmentation spectra to identify a CSM.
  • MS2_MS3: Specifies the MS2_MS3 mode of acquisition, which is used only for cleavable crosslinkers. The mass spectrometer first fragments every precursor ion, usually by CID with lower energy. This fragmentation cleaves the crosslinker and generates the signature fragment ions. Then the mass spectrometer uses MS3 CID or IT HCD for up to four corresponding spectra, where it fragments the peptide backbone.
  • NonCleavable: Uses the standard enumeration of all possible peptide pairs to identify the crosslinker. The maximum size of the database FASTA file is 400 proteins. This mode is used for non-cleavable crosslinkers.
  • Noncleavable_open: Uses the sequence tags, immonium ions and diagnostic ions from the fragmentation spectrum to make an initial prediction for peptide sequences involved in the crosslinked peptide pair in combination with the standard enumeration approach. The Noncleavable_open search algorithm is the fastest non-cleavable crosslinker search for databases up to 5000 proteins.

Crosslink Modification

Specifies the crosslink modification used for linking the proteins.

If no modification is available, define the crosslink modification in the Chemical Modifications view, including the settings of the crosslink modification in the Modification Extended Properties dialog box. (See Adding crosslinkers.)

Minimum S/N (Input Data)

Specifies the signal-to-noise spectrum threshold of the MS1 precursor.

Default: 1.5

Enable protein N-terminus linkage

Specifies the crosslink modification used for linking the proteins.

Specifies if a crosslink attachment to the n-terminus of the protein is allowed.

Default: false

SequenceTag Min Length

The minimum length of a sequence tag to consider.

Range: 1–100; default: 3

SequenceTag Max Length

The maximum length of a sequence tag to consider.

Range: 1–100; default: 9

SequenceTag Score Filter

Filters out bad sequence tags based on the score when the Noncleavable_open method is used.

Default: false

Minimum S/N (Sequence Tags)

The minimum signal-to-noise accepted for detecting sequence tags when the Noncleavable_open method is used.

Default: 10

Filter immonium ions

Removes immonium ions prior to sequence tag detection when the Noncleavable_open method is used.

Default: true

Filter duplicate ions

Removes indistinguishable peaks prior to sequence tag detection when the Noncleavable_open method is used.

Default: true