The Hi Res. Feature Detector node uses the sliding window and Xtract algorithms to perform spectral deconvolution and measure all of the deconvoluted features and their quantitation traces. The parameters used in this node are essential to the LFQ workflow. Set them according to the data you are analyzing.

One of the key parameters is the Sliding Window Averaging Width RT. This parameter establishes the RT for the sliding window average. Set this parameter to match the full width at half max of a representative peak from the analyzed data. It is recommended to measure the peak width of an average feature in your data set. Do not choose the most abundant peak or one that is at noise level.

The best parameter setting is based on your chromatography:

  • If the window is too wide, then low intensity signals are averaged out of the data.
  • If the window is too narrow, then you lose the advantages of signal averaging.

The following table lists the node parameters.

Table ProSightPD Hi Res. Feature Detector parameters

Parameter

Description

1. Xtract

S/N Threshold

Specifies the minimum signal-to-noise ratio for data that the node analyses.

Lowest m/z

Specifies the portion of the input spectrum that the Xtract algorithm processes.

Min: Specifies the lowest end of the input spectrum.

Highest m/z

Specifies the portion of the input spectrum that the Xtract algorithm processes.

Max: Specifies the highest end of the input spectrum.

Min Precursor Mass

The minimum precursor mass that the Xtract algorithm considers.

Max Precursor Mass

The maximum precursor mass that the Xtract algorithm considers.

Lowest Charge

Sets the low end of the allowable range for the number of charge states that must appear for a component to be recognized. The Xtract algorithm rejects potential components with fewer than the minimum or greater than the maximum number of charge states.

Highest Charge

Sets the high end of the allowable range.

Minimum Number of Detected Charge States

Minimum required number of observed charge states for data to be analyzed by the Xtract algorithm.

For top-down data, the recommended minimum is 3. Some very low-abundance species might have only 2 observable (greater than noise) peaks. However, setting this value to less than 3 lets noise through and results in false positives.

Relative Abundance Threshold

Specifies a threshold below which the node filters out data for data reporting. This option sets a relative threshold as a percentage of the most abundant component in the spectrum. The most abundant peak in the deconvolved spectrum has a relative abundance of 100 percent, and all other peaks are calculated relative to that one. For example, if the highest peak has an absolute abundance of 1000, the relative abundance is 1 percent, and no peaks below an absolute abundance of 10 appear in the deconvolved spectrum.

Range: 0–100

Zero (0) displays all results; 100 displays only the most abundant component.

Resolution at m/z 400

Defines the resolution of the source spectrum at an m/z value of 400.

Fit Factor

Measures the quality of the match between a measured isotope pattern and an averagine distribution of the same mass. Enter a value between 0 and 100%

  • 0% requires a low fit only.
  • 100% means that the measured isotope profile is identical to the theoretical averagine isotope distribution.

Remainder Threshold (%)

Specifies the height of the smaller overlapping isotopic cluster, as a percentage, with respect to the height of the most abundant isotopic cluster when the Xtract algorithm attempts to resolve overlapping isotopic clusters.

For example, if one isotopic cluster in a spectrum has an abundance of 100, and you set the Remainder Threshold parameter to 30%, the Xtract algorithm ignores any overlapping clusters with an abundance less than 30.

Min. Intensity

Specifies a minimum intensity threshold to filter out possible background noise, including when you set the S/N Threshold parameter to zero.

Expected Intensity Err.

Specifies the permissible percentage of error allowed in calculating the ratio of the most abundant isotope to the next isotope higher in mass in the isotope series.

Consider Overlaps

When selected (default), indicates that the Xtract algorithm is more tolerant of errors when the spectrum intensity is significantly higher than expected for the theoretical isotopic cluster.

This option can lead to increased false positives; select it only when you expect overlapping isotopic clusters in a data set.

2. Sliding Window Parameters

Averaging Width RT

Specifies the retention time, or the width, of the sliding window, in minutes.

  • Reducing this value improves time resolution but reduces execution speed and possibly sensitivity.
  • Increasing this value increases execution speed but reduces time resolution and might increase sensitivity.

You can achieve the best results when the window width is between one-quarter and twice the width of the characteristic peaks in the spectrum. For most uses, the optimum value might be half the width of the characteristic peaks. For example, if those peaks have a width of one minute, the optimum width is 0.5 minutes.

Offset Type

Specifies the offset between successive sliding windows as a number of scans or as a percentage value. Select from:

  • Scan—Uses the Offset Scan setting
  • Percent—Uses the Offset Percentage setting

Offset Scan

Offsets each window from its predecessor by the user-specified number of scans. An offset of n means that each window begins n scans after the beginning of its predecessor.

Offset Percentage

Offsets each window from its predecessor by the user-specified percentage of the window width.

  • An offset of 30% causes each window to begin 30% after the beginning of its predecessor and overlap the last 70%.
  • An offset of 100% causes successive windows to be adjacent with no overlap.

Merge Tolerance

Determines how close two components in successive sliding windows must be in mass for the application to identify them as a single component.

  • A value that is too high might result in merging components that should remain separate.
  • A value that is too low might result in false positives when components remain separate that should have merged.

Merge Mode

Determines how spectra are merged:

  • Batch—Merges spectra as a batch, which minimizes the effect of scan to scan variation (Recommended).
  • Incremental—Merges spectra incrementally, one-by-one, making the result sensitive to scan-by-scan variations for long eluting species (Legacy).

Minimum Number of Charge States Observed

Minimum required number of observed charge states for data to be analyzed by kDecon.

For top-down data, the recommended minimum is 3. Some very low-abundance species might have only 2 observable (greater than noise) peaks. However, setting this value less than 3 lets noise through and result in false positives.

Minimum Number of Sliding Window Detections

Specifies the minimum number of sliding window intervals that a component must appear in for the application to consider the component valid.

Set this parameter to a value large enough to exclude results that are implausibly narrow in retention time but small enough to include results of realistic duration. Values in the range of 3 through 8 generally give good results. A good approach is to use whichever is larger: 3 or the minimum number of windows that can fit into a peak.

  • A value that is too low might allow noise peaks to appear as false positives.
  • A value that is too high might result in legitimate components being discarded.

Biggest Gap Type

Specifies the maximum allowed separation in retention time between two successive individual members of a merged component identified by the sliding windows algorithm, in minutes.

  • Scans
  • Retention time

Biggest Gap Scans

Specifies the greatest number of scans between subsequent feature detection for grouping similar features.

  • If the separation exceeds this value, the application divides the candidate component into two merged components separated by a gap in retention time. A value that is too high for this parameter might result in merging components that should remain separate.
  • A value that is too low might result in false positives when components remain separate that should have merged.

Set this parameter comparable to or slightly less than the expected separation in retention time between distinct components with the same mass.

Biggest Gap Retention Time

Specifies the greatest amount of time between feature detection events for grouping similar features.

3. Feature /PrSM Connection

Mass Tolerance

Delta mass (in Daltons) allowed for mapping PrSMs to features.

RT Threshold

Retention time threshold for mapping PrSMs to features:

  • Minimum value = 0
  • Maximum value = (unchecked)

4. Feature/Trace Connection

Trace Mass Tolerance

Specifies the trace tolerance.

  • Minimum value = 0.01 Da 0.1 ppm
  • Maximum value = (unchecked)

Number Of Smoothing Points

  • Minimum value = 0
  • Maximum value = (unchecked)

Time Range (min)

  • Minimum value = 0
  • Maximum value = (unchecked)

Trace Smoothing Type

Determines the method to smooth the traces:

  • Gauss
  • MovingMean (Default)
  • None

5. Multithreading Options

CPU Usage

Determines the amount of CPU to direct toward processing:

  • High—All available threads less 1 (Default)
  • Medium—50% of the available threads
  • Low—1 thread