Use the settings of the nodes’ Normalization Mode parameter to specify how they perform normalization:

  • (Default) None: Does not perform normalization.
  • Total Peptide Amount: Sums the peptide group abundances for each sample and determines the maximum sum for all files. The normalization factor is the factor of the sum of the sample and the maximum sum in all files.
  • Specific Protein Amount: Calculates the normalization factor from the abundances of selected proteins in the specified FASTA file. You can specify a FASTA file (use the Proteins for Normalization parameter to specify the name of the FASTA file) that can contain one or multiple proteins. The Reporter Ions Quantifier node and the Precursor Ions Quantifier node use all proteins in the FASTA file that are contained in the result file and that have any protein abundance. This setting calculates the maximum sum for all files. The normalization factor is the factor of the sum of a sample and the maximum sum in all files.
  • RT Dependent: This method corrects for datasets where different normalization factors would be required for different retention time ranges within the files. In this mode, the normalization is performed RT dependent by aligning sample abundances of consensus features from all peptides to the median abundance of all samples. This option is not supported for fractioned samples.

If you set the Normalization Mode parameter to Specific Protein Amount but do not select a FASTA file, or if the result file contains no proteins that appear in the FASTA file, the nodes cannot perform normalization.

When normalization is based on specific proteins, the Reporter Ions Quantifier node and the Precursor Ions Quantifier node sum the abundance values of all proteins in the result file that reside in the selected FASTA file. They compare the protein sequences in the FASTA file to the sequences in the result file rather than to the accession strings. You can therefore use the same FASTA file when the title line is slightly different.

After calculating the normalization factors, the Reporter Ions Quantifier node and the Precursor Ions Quantifier node normalize quan spectra (for reporter ion quantification) or consensus features (for label-free and precursor ion quantification) by dividing abundances with the normalization factor over all samples.