Fraction variable in PD annotation

[Tahyne]

Hello, I am facing the following issue:
I have an experiment of TMTpro 16 plex in 4 runs with 58 samples + 6 (internal control). The experiment was fractionated in gradient length (table and experiment information attached).
In order to create the AnnotationPD file should I consider fractions with the same values or not? In that case, which one is more adequate to use (same fractions values or different ones)? And what should be the impact if I use both cases to compare?

TMT16_HiRIEF_Materials and Methods.pdf

[mstaniak]

Hi,
how many mixtures are there? As many as unique values in the "gradient length" column?
Fraction column in annotation can be the same as the "Sample" column.

[Tahyne]

There are 4 mixtures