# Spectrum Count Normalization in Scaffold

The following document describes the procedure for normalizing protein spectrum counts in Scaffold. It also includes an example calculation.

Normalization Procedure

The normalization process involves three steps:

1. Calculate the total number of spectra in each BioSample
2. Calculate the average number of spectra across all BioSamples
3. Multiply each spectrum count in each sample by the average count over the BioSample's total spectrum count

Example Calculation

Suppose we had the following results in Scaffold.

 BioSample 1 BioSample 2 Protein A 12 Protein A 8 Protein B 6 Protein B 3 Protein C 4 Protein C 3 Total 22 Total 14

We start by finding the total number of spectra in each BioSample. BioSample 1 has a total of 22 spectra and BioSample 2 has a total of 14 spectra. Next, we calculate the average number of spectra across all BioSamples. In this example the average is 18. Scaffold then multiplies each spectrum count by the average over the total for each BioSample. Counts in BioSample 1 will be multiplied by (18/22) and those in BioSample 2 will be multiplied by (18/14).

 BioSample 1 (Normalized) BioSample 2 (Normalized) Protein A 10 Protein A 10 Protein B 5 Protein B 4 Protein C 3 Protein C 4