How to analyse several separate DIA files together as a single file

[Sadegh1994]

Dear all

I am seeking your expertise in analyzing my proteomics data. To enhance the coverage of my dataset, I performed Ph fractionation and subsequently injected the fractions separately. As a result, I have several seperate raw files that collectively represent a single sample.

Since I had run DIA approach, I would like guidance on how to merge these separate files into one comprehensive file and that can be used for comparative analysis between the control and treatment groups. Does DiaNN has this option or u suggest to use another software for data analysis, if yes please give me a little details about it.

Thank you in advance for your assistance.

Best regards,
Sadegh

[vdemichev]

Hi Sadegh,

I assume you have multiple samples, each measured in multiple fractions, and you'd like to compare protein intensities in those samples? In general, fractionation is not the best for quantitative comparisons, unless you have an internal standard that would allow you to account for the technical variation introduced by fractionation. If you don't have this, I would suggest to try:

• For each protein, define protein quantity = maximum of all precursor quantities across all fractions in the sample and precursors matched to the protein.
• Alternatively, for each precursor sum its levels across the fractions, then define protein quantity in the sample as the summed intensity of the precursor for which this summed intensity is the highest among all precursors matched to the protein.

With regard to using DIA-NN, makes sense to analyse the experiment with normalisation disabled. Can then perform normalisation using a custom script in R once proteins are quantified.

Best, Vadim