Normalization Methods


Skyline offers many different ways to perform normalization.

These are normalization options that may be available at "Settings > Peptide Settings > Quantification":

  • None

    The normalized area is the sum of the transition peak areas of all of the transitions whose Isotope Label Type is not an Internal Standard Type specified at "Settings > Peptide Settings > Modifications"

  • Equalize Medians

    The unnormalized area is divided by the median transition peak area in the result file and multiplied by the median of the median peak areas across all of the result files. The median peak area for each result file can be found in the "Median Peak Area" column in the Document Grid. The peak areas that contribute to the median are:

    • The peak area of every MS2 transition
    • The Total Area MS1 of every precursor
  • Ratio to Global Standards

    The unnormalized area is divided by the sum of the peak areas in the Result File for all of the peptides or molecules whose Standard Type is "Global Standard". If the "Explicit Global Standard Area" value has been set in the Document Grid then that value is used instead of the actual sum of peak areas

  • Total Ion Current

    The unnormalized area is divided by the Total Ion Current Area for the Result File. When extracting chromatograms from full spectrum data, Skyline sums the intensities in the points that correspond to MS1 spectra in the Total Ion Current chromatogram.

  • Ratio to <Isotope Label Type>

    The sum of the transition peak areas from the other isotope label type is divided by the sum of the transition peak areas from the normalization isotope label type.

    The behavior of this normalization method depends on whether "Simple precursor ratios" is checked at "Settings > Peptide Settings > Quantification". If "Simple precursor ratios" is checked then all of the transitions contribute to the peak area sums. If "Simple precursor ratios" is unchecked then Skyline matches transitions between the two label types and only transitions which can have a peak area in both label types are included.

In addition, the normalization method for a particular peptide or molecule can be overridden by setting the value in the "Normalization Method" column in the Document Grid. In that column one can also choose the normalization method "Ratio to Surrogate <XXXX>". For more information see the Surrogate Standards tips page.

When defining a Group Comparison or when looking at the Peak Area graph in Skyline one can choose the normalization method "Default". This normalization method means to apply the default normalization method but also includes the MS Level that might have been chosen at "Settings > Peptide Settings > Quantification".

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