Table of Contents

guest
2023-02-07
   Quantification
     Batch Calibration
     SureQuant and Triggered Acquisition
     Surrogate Standards
     How Skyline Calculates Peak Areas and Heights

Quantification


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.




SureQuant and Triggered Acquisition


SureQuant Tutorial (posted May 9, 2022)

To learn more about using Skyline with SureQuant take a look at this tutorial. Here is the supplemental data for the tutorial.

Triggered Acquisition Setting

As of Skyline-Daily 20.1.1.83, 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


Definitions:

  1. Background height: minimum intensity at peak boundaries (the blue line in the image below)
  2. Background area ("Background" in the table below): total integrated area of the minimum of background height and intensity at each point
  3. Peak height ("Height" in the table below): maximum intensity between peak boundaries minus background height
  4. Peak area ("Area" in the table below): total integrated area within peak boundaries minus background area. Area plus background always equals raw area below the curve.

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:

 

The background area is the rectangle bounded by the background height (blue "125" line) and peak boundaries (gray dotted lines), less the areas of the chromatogram that descend below the background height (light blue areas). You may have noticed that the light blue area at RT=14.55 does not exactly match the shape of the chromatogram: for speed and simplicity the area calculation simply uses the RT of the points to left and right rather than calculating the slope and intercept to get the RT where the chromatogram nominally crosses the background height line. This is a very small discrepancy in most cases.

Skyline graphs this background area information when you select a single transition. You will see the peak area value shaded in red, and the background area shaded in gray. You can place the cursor to the left of the y-axis and use the scroll wheel to zoom in the y-dimension to better inspect the background shading.