The Xtract Node is available only if you have a valid Xtract license.
The Xtract node deconvolutes the precursor ions for all high-resolution data, regardless of the fragmentation type.
Electrospray ionization (ESI) of peptides and proteins produces a series of multiply charged ions with an associated mass-to-charge (m/z) ratio. The resulting mass spectra are crowded by these multiple charge values for each molecular mass and an isotopic cluster for each nominal m/z value. Xtract simultaneously combines all observed charged states into a single isotopic cluster with a charge of +1 (a process called deconvolution) and collapses ESI mass spectra isotope patterns into a single peak from complex peptide samples on the basis of their mass-dependent isotopic mean pattern. All signals corresponding to one peptide in the sample are reduced to one singly charged monoisotopic peak, significantly reducing the number of signals, increasing the signal intensity, and improving the signal-to-noise ratio. You can directly use the mass list produced for database searching.
The results of the deconvoluted fragment spectra (or extracted spectra) are used as the input for the search engines. In a simple SEQUEST search, the selected .raw file is passed to the Spectrum Selector node, which passes qualifying MS/MS scans based on the specified criteria to Xtract and finally on to the search engines. In this type of workflow, the Xtract node deconvolutes the input m/z spectra to singly charged (MH+) mass peak lists.
The extracted MS/MS spectra, not the MS/MS spectra that were acquired by the instrument, are used in the peptide search.
When used properly, this processing tool provides a cleaner fragmentation spectrum with peaks of only +1 charge states to be submitted to the search engine. It can effectively increase the confidence of the peptide matches by reducing matches that occur randomly due to spectrum noise.
The Xtract node only works with high-resolution spectral data.
The Xtract node is only available through the Workflow Editor.
The Xtract node has several parameters that could change the output. You might need to investigate and test which settings are best for your data. When you start your investigation, use the default values. With the application, investigate the effect of applying deconvolution with different parameter settings before searching for peptides by either performing different searches and comparing the results in a multiconsensus report, or by creating a workflow with multiple search branches.
By default, Xtract outputs full isotope patterns in the deconvoluted spectrum. To change this behavior, set the Monoisotopic Mass Only parameter to True.
Parameters | Description |
|---|---|
Highest Charge | Specifies the highest charge state that you can set for the deconvolution of multiply charged data. Range: 0–30 Default: 20 |
Highest m/z | Specifies the highest mass-to-charge value for spectral peaks in the measured spectrum that are considered for Xtract. Range: 0–no maximum; default: 2000 Da |
Lowest Charge | Specifies the lowest charge state that you can have for the deconvolution of multiply charged data. Range: 0–30; default: 1 |
Lowest m/z | Specifies the lowest mass-to-charge value for spectral peaks in the measured spectrum that are considered for Xtract. Range: 0–no maximum; default: 200 Da |
MonoIsotopic Mass Only | Determines whether the isotopic pattern—that is, all isotopes of a mass—is removed from the spectrum.
|
Overlapping Remainder | Specifies the fraction of the more abundant peak that an overlapping multiplet must exceed in order to be processed (deconvoluted). If an isotopic envelope falls below this fraction, it is not processed. This parameter concerns data in which more than one isotopic envelope overlaps in a spectrum. Range: 0.0–1.0; default: 0.25 |
Required Fitting Accuracy | Specifies the accuracy required for a pattern fit to be considered valid. This parameter refers to the degree to which the experimentally derived isotopic envelope should resemble the theoretical isotopic pattern for an average envelope of that mass. Range: 0.0–1.0; default: 0.44 |
Resolution@400 | Defines the resolution at 400 m/z. This parameter is not needed if FTMS data is extracted because the data contains the information in the spectrum. You must set this parameter for all other spectrum types. Range: 7000–no maximum; default: 100 000 |
S/N Threshold | Specifies a signal-to-noise (S/N) threshold above which Xtract processes a peak. Any spectral peak must be x times the intensity of the calculated noise for that spectrum before Xtract considers it. Range: 0–no maximum; default: 3 |