The Non-Fragment Filter node removes precursor artifacts from the MS/MS input spectra. Examples of these artifacts are the peaks of unfragmented precursor ions, peaks of charged-reduced species of precursor ions and their resulting neutral loss peaks, and precursor artifact peaks due to higher harmonic frequencies (overtones). ETD and ECD fragment spectra typically include two types of ion peaks: true fragment ion peaks and peaks that are related to the precursor ions. Figure 355 gives an example of a spectrum that contains these two types of ion peaks.

The Non-Fragment Filter node calculates and removes non-fragment ion peaks from the mass lists that are generated before submission to database searches. Non-fragment ion peaks can lead to false positive identifications in database searches that use search algorithms that score the experimentally generated spectrum versus a calculated theoretical spectrum. The removed peaks are not likely to be fragment peaks from ETD or ECD data. In Figure 355, this node is restricted to precursor ion peaks present in ETD and ECD spectra.

Example of a typical ECD spectrum (BSA, peptide YICDNDTISSK)
Example of a typical ECD spectrum (BSA, peptide YICDNDTISSK)

For information about adding a non-fragment filter node for Orbitrap or FT ICR data, see Correct workflow errors.

Given the mass of the precursor, you can calculate the theoretical mass-to-charge ratio (m/z) values of these non-fragment peaks and remove every peak within these regions with a certain mass tolerance.

Calculate these mass windows as follows:

  • Precursor peaks: Calculate the final mass window used to remove precursor peaks as follows:
  • window_lower_mass = precursor_m/z - offset
  • window_upper_mass = precursor_m/z + (#isotope_peaks/precursor_charge) + offset
  • The application looks up the number of isotope peaks (#isotope_peaks) in the static table according to the uncharged mass.
Uncharged mass and number of isotope peaks used in calculating precursor peaks

Uncharged mass

Isotope peaks

< 1000

3

1000 – 2000

4

2000 – 3000

5

3000 – 4000

6

4000 – 5000

7

5000 – 6000

8

6000 – 7000

9

> 7000

10

  • Precursor overtone peaks: Calculate the final mass window used to remove precursor overtone peaks as follows:
  • window_lower_mass = overtone_m/z - offset
  • window_upper_mass = overtone_m/z + (#isotope_peaks/precursor_charge) + offset
  • where the overtone_m/z is at 1/2, 1/3, 1/4 of the precursor mass-to-charge ratio (m/z).
  • Charge-reduced precursor peaks: Calculate the final mass window used to remove charge-reduced precursor peaks as follows:
  • window_lower_mass = charge_reduced_precursor_m/z - (proton_mass/charge) - offset
  • window_upper_mass = charge_reduced_precursor_m/z + (#isotope_peaks/charge) + offset
  • where charge is the charge of the reduced species. Charge_reduced_precursor_m/z is calculated by adding electrons to the precursor until charge 1 is reached but without removing protons. For example, if the precursor ion is [M+4H]4+, the next charge-reduced ion is [M+4H]3+, then [M+4H]2+, and so forth.
  • Neutral loss peaks of charge-reduced precursor ions: The final mass window used to remove neutral loss peaks of charge-reduced precursor ions depends on whether all peaks within a large neutral loss mass window should be removed or only the peaks within smaller windows around the positions of known neutral losses from charge-reduced species.
  • When you remove only small windows around known neutral losses, calculate the used mass windows as follows:
  • window_lower_mass = neutral_loss_m/z - offset
  • window_upper_mass = neutral_loss_m/z + (#isotope_peaks/charge) + offset
  • where charge is the charge of the reduced species.
  • If you must remove a large mass range to eliminate neutral loss peaks, calculate the used mass window as follows:
  • window_lower_mass = charge_reduced_precursor_m/z - (maximum_loss_mass/ charge)
  • window_upper_mass = charge_reduced_precursor_m/z
  • If you must remove only known neutral loss masses, use the values in the following table.
Neutral loss masses

Mass

Neutral loss

17.027 Da

NH3

18.011 Da

H20

27.995 Da

CO

32.026 Da

CH3OH

34.053 Da

N2H6 (2xNH3)

35.037 Da

H4NO

36.021 Da

H4O2 (2xH2O)

44.037 Da

CH4N2

45.021 Da

CH3NO

46.006 Da

CH2O2

46.042 Da

C2H6O

59.037 Da

C2H5NO

59.048 Da

CH5N3

73.089 Da

C4H11N

74.019 Da

C3H6S

82.053 Da

C4H6N2

86.072 Da

C3H8N3

99.068 Da

C4H9N3

101.095 Da

C4H11N3

108.058 Da

C7H8O

131.074 Da

C9H9N

The main purpose of the Non-Fragment Filter node is to remove precursor peaks from the spectra that are not related to peptide fragments and can therefore increase the risk of the search engines making false positive matches. If you add a Non-Fragment Filter node to the workflow for processing data taken from Orbitrap instruments, Thermo Fisher Scientific recommends that you remove most of the precursor peaks. Setting the window to a smaller width increases the risk of leaving some of the precursor peaks or their side bands in the spectrum. The following figure shows the recommended settings with wider tolerances.

Non-Fragment Filter node settings for data taken from LTQ Orbitrap instruments
Non-Fragment Filter node settings for data taken from LTQ Orbitrap instruments

Peaks arising from overtones are rarely seen within Orbitrap spectra but are prominent peaks in spectra from the LTQ-FT instruments. The range in which neutral loss peaks from the charge-reduced precursor peaks are removed is scaled by the charge of the charge-reduced peak. Therefore, if you specify a value of 130 Da, the application removes neutral loss peaks within a 130-Da range for +1 peaks, a 65-Da range for +2 peaks, and so forth. To remove neutral losses, you can remove either every peak within the specified range or only those peaks from an internal table of known neutral loss masses from charge-reduced precursor ions, such as those shown in the table below.

Mass of known neutral losses from charge-reduced precursor ions (Sheet 2 of 2)

Mass

Neutral loss

17.027

NH3

18.011

H2O

27.995 Da

CO

32.026 Da

CH3OH

34.053 Da

N2H6 (2xNH3)

35.037 Da

H4NO

36.021 Da

H4O2 (2xH20)

44.037 Da

CH4N2

45.021 Da

CH3NO

46.006 Da

CH202

46.042 Da

C2H6O

59.037 Da

C2H5NO

59.048 Da

CH5N3

73.089 Da

C4H11N

74.019 Da

C3H6S

82.053 Da

C4H6N2

86.072 Da

C3H8N3

99.068 Da

C4H9N3

101.095 Da

C4H11N3

108.58 Da

C7H8O

131.074 Da

C9H9N

The following table describes the parameters for the Non-Fragment Filter node.

Non-Fragment Filter node parameters

Parameters

Description

Remove Precursor Peak

Determines whether to remove precursor artifact peaks from the MS/MS input spectra. The application removes all peaks within a given mass window around the precursor mass-to-charge ratio.

  • (Default) True: Removes precursor artifact peaks from the MS/MS input spectra.
  • False: Does not remove precursor artifact peaks from the MS/MS input spectra.

Mass Window Offset

Specifies the size of the mass-to-charge ratio (m/z) window, in daltons, from which the Remove Precursor Peak option removes precursor artifact peaks from the spectrum.

Range: 0.0 Da–no maximum; default: 1.0 Da

Remove Charge Reduced Precursors

Determines whether to remove the charge-reduced precursor peaks found in an spectrum. These precursors only complicate the analysis of the fragment spectra. The application removes all peaks within a given mass window around the precursor mass-to-charge ratio.

  • (Default) True: Removes the charge-reduced precursor peaks found in a spectrum.
  • False: Does not remove the charge-reduced precursor peaks found in a spectrum.

Mass Window Offset

Specifies the size of the mass-to-charge ratio (m/z) window, in daltons, from which the Remove Charge Reduced Precursors option removes charge-reduced precursor peaks.

Range: 0.0 Da–no maximum; default: 0.5 Da

Remove Neutral Loss Peaks

Determines whether to remove neutral loss peaks from the spectra. Charge-reduced species formed in reactions can undergo neutral loss reactions, creating non-fragment peaks that complicate the analysis by typical search engines. The application removes all peaks within a given mass window around the precursor mass-to-charge ratio.

  • (Default) True: Removes neutral loss peaks from the spectra.
  • False: Does not remove neutral loss peaks from the spectra.

Mass Window Offset

Specifies the tolerance for the Remove Neutral Loss Peaks option, in daltons.

Range: 0.0 Da–no maximum; default: 0.5 Da

Remove Only Known Masses

Determines whether to remove overtone peaks from LTQ FT or LTQ FT Ultra spectra. The application removes all peaks within a given mass window around the precursor mass-to-charge ratio.

  • (Default) True: Removes overtone peaks from LTQ FT or LTQ FT Ultra spectra.
  • False: Does not remove overtone peaks from LTQ FT or LTQ FT Ultra spectra.

Maximum Neutral Loss Mass

Specifies the maximum allowed neutral loss.

Range: 0.0 Da–no maximum; default: 120 Da

Remove Precursor Overtones

Determines whether to remove precursor overtone peaks in the spectrum from the input spectrum. Overtones, or harmonics, are an artifact of FT detectors. The application removes all peaks within a given mass window around the precursor mass-to-charge ratio.

  • (Default) True: Removes precursor overtone peaks from the input spectrum.
  • False: Does not remove precursor overtone peaks from the input spectrum.

Mass Window Offset

Specifies the size of the mass-to-charge ratio (m/z) window, in daltons, from which the Remove Precursor Overtones option removes precursor overtone peaks.

Range: 0.0 Da–no maximum; default: 0.5 Da

Remove TMT Tag Ions

Determines whether to remove the peaks that are known TMT reporter ions, the peak from the intact tag ion, and the charge-reduced losses of the reporter tags of the precursor from the spectrum.

  • True: Removes the peaks that are known TMT reporter ions, the peak from the intact tag ion, and the charge-reduced losses of the reporter tags of the precursor from the spectrum.
  • (Default) False: Does not removes the peaks that are known TMT reporter ions, the peak from the intact tag ion, and the charge-reduced losses of the reporter tags of the precursor from the spectrum.

Mass Window Offset

Specifies an additional offset to apply to each mass window. For the TMT reporter tags and the intact tag ion, the offset defines the mass window in which all peaks are removed from the spectrum. For the charge-reduced reporter tag losses, the mass window spans from the monoisotopic peak to the (assumed) last peak of the corresponding isotope pattern.

Default: 0.5 Da

Remove Specified Masses

Activates the Edit Parameter Text for Remove Specified Masses dialog box so that you can enter the masses of the peaks to remove. When it removes the peaks, the application removes all peaks within a given mass window around the precursor m/z.

Separate items by a semicolon (;), or place each entry on a separate line in the dialog box.

Remove Specified Delta Masses

Activates the Edit Parameter Text for Remove Specified Delta Masses dialog box so that you can enter the peaks with a given delta mass of the precursor ion to remove. When it removes the peaks, the application removes all peaks within a given mass window around the precursor m/z.

Mass Window Offset

Specifies the size of an additional offset to apply to each mass window. For the Remove Specified Masses parameters, the mass window spans around the monoisotopic mass. For the Remove Specified Delta Masses parameter, the mass window spans from the monoisotopic peak to the (assumed) last peak of the corresponding isotope pattern.

Default: 0.5 Da