Table of Contents

     Batch Calibration
     Triggered Acquisition in Skyline 20.2
     Surrogate Standards
     How Skyline Calculates Peak Areas and Heights


Calibrated quantification (a.k.a. absolute quantification) has been added to Skyline in version 3.5. We plan on extending documentation of this feature in the future in a number of ways:

  1. Updating the existing Absolute Quantification tutorial
  2. Writing a new tutorial on calibrated quantification that covers all of the available applications
  3. Including a detailed demonstration of the functionality in a future webinar

Until this work can be completed, however, you can find attached to this page a set of PowerPoint slides which hopefully provide enough of a rough overview of what is now possible that anyone interested can at least get started with the new functionality.

Also, the Skyline Tutorial Webinar #12 gives some initial coverage on this feature near the end of the recording (and in presentation slides).

Batch Calibration

Skyline 19.1 allows you to specify the "Batch Name" on replicates so that different replicates will use a different set of external standards.

See the attached PowerPoint to see how to use this feature.

Triggered Acquisition in Skyline 20.2

As of Skyline-Daily, Skyline-Daily has support for "Triggered Acquisition" methods.

A Triggered Acquisition method is one where the mass spectrometer has been told to begin collecting MS2 scans for one analyte when the mass spectrometer sees particular transitions for a different precursor.

This will enable Skyline to work better with assays such as Thermo's SureQuant Targeted Mass Spec Assay Kits

The Transition Settings > Instrument tab will have a "Triggered Acquisition" checkbox which tells Skyline that there may be large gaps between the points in an analyte's chromatograms.

When Triggered Acquisition is selected, Skyline will detect these gaps and make sure that integration boundaries do not cross these gaps. Also, Skyline will perform no background subtraction when Triggered Acquisition is enabled.

For more information, see the attached PowerPoint.

Surrogate Standards

Sometimes you want to normalize a particular analyte against a different molecule. Skyline supports this with the "Surrogate Standard" feature.

To designate that a molecule can be used as a surrogate standard, right click on the molecule in the Targets tree and choose "Set Standard Type > Surrogate Standard".

You have to use the Document Grid to change the normalization method of the analyte. The "Normalization Method" column is not shown by default, so you need to customize a view in the Document Grid and add that column. You can start by choosing "Peptides" from the Reports dropdown on the Document Grid. Then, choose "Customize Report" and add the "Normalization Method" to the view. The Normalization Method column is under "Proteins > Peptides". (also note the button at the top with the binoculars icon can be used to find columns by name)

If you have surrogate standards in your document, then the "Normalization Method" column will have options of the form "Ratio to surrogate..."

How Skyline Calculates Peak Areas and Heights


  1. Background height: minimum intensity at peak boundaries
  2. Background area (named Background):
    • rectangular area of background height times the length between peak boundaries (pre-v1.4 patch 1)
    • total integrated area of the minimum of background height and intensity at each point (v1.4 patch 1 and later)
      (Note: the unit of RT time is second for this calculation)
  3. Peak height (named Height): maximum intensity between peak boundaries minus background height
  4. Peak area (named Area): total integrated area within peak boundaries minus background area (Note: the unit of RT time is second for this calculation)
    • area can be zero and background greater than total raw area (pre-v1.4 patch 1)
    • area plus background always equals raw area (v1.4 patch 1 and later)

NOTE: Skyline uses points that have been linear interpolated from the raw data onto a uniform interval over the duration of the chromatogram in detecting its peak boundaries and calculating its peak areas. These are also the points Skyline displays in its chromatogram graphs. Skyline uses several types of smoothing (1st derivative, 2nd derivative and Savitzky-Golay) in order to place its automatically calculated peak boundaries. These smoothed curves are available for display in the Skyline chromatogram graphs. Skyline does not, however, use smoothed data in calculating peak areas (or area under the curve - AUC). It always uses the raw interpolated points presented in the unsmoothed graphs.

Example of calculation of peak height and background area:


Note: with v1.4 patch 1 and higher the light blue areas are not included in the background area.