Increase number of peptides needed for Quant

[JLewisMicro]

Hi,

We're analysing some bacterial proteome data between a mutant and wildtype strain on FragPipe v18 and we're having an issue with a protein being quantified in the mutant strain based on a single peptide with a low hyperscore (the peptide is sequence is completely absent in the mutant). Is there a straightforward way to restrict the number of peptides needed for quant to 2 peptides?

Thanks, Jessica

[fcyu]

Hi Jessica,

We can't control the min number of peptide sequences, but we can control the min number of peptide ions (peptidoform+ charge) in IonQuant:

Best,

Fengchao

[JLewisMicro]

log_2022-06-28_17-33-32.txt

Hi Fengchao,

Thanks for the suggestion. I changed that setting yesterday to 3 and it did not filter out the data (all other settings were the same as the original run), and I've included a snapshot of the original data, with 2 or less identifications in the mutants and 8 or more in the wildtype strain (protein highlighted in yellow), and the log from my run yesterday with the minions increased to 3. Is this a bug in FragPipe?

Thanks, Jessica

[fcyu]

Hi Jesscia,

What you showed is the spectral count, not the peptide ion counts. And, that parameter is used for label-free quantification. So, for those proteins with less than 4 peptide ions, the intensities will be 0. But the protein will still be listed as being identified.

Best,

Fengchao

[JLewisMicro]

Hi Fengchao,

Where can I find the number of peptide ion counts? I've looked in some of the different output files and I can't find them. I'm happy with the intensities being 0 and th eproteins still being marked as identified.

Thank you for you help with this, Jessica

[fcyu]

I don't think they are in the file. I will add them in the future.

Best,

Fengchao

[JLewisMicro]

Hi Fengchao,

I've gone back through my previous run, and adjusting the mio ions to 2 did remove the protein in the MaxLFQ intensity, but not the standard intensity.

Is there a setting that can be altered to have the MaxLFQ intensity values as the output in the reprint.int.tsv, rather than the stendard intensity?

Thanks, Jessica

[fcyu]

For the reprint.int.tsv, we write the intensity from top-N algorithm, so, there is no min ion settings.

Best,

Fengchao

[anesvi]

I did quite a bit of testing myself and top-N ( with large N, meaning all peptides) is better than maxLFQ intensity when using aS input to SAINTexpress scoring in reprint-apms. This is why we write that column to reprint.Int.tsv file

Alexey

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From: JLewisMicro @.> Sent: Tuesday, June 28, 2022 9:37:55 PM To: Nesvilab/FragPipe @.> Cc: Subscribed @.***> Subject: Re: [Nesvilab/FragPipe] Increase number of peptides needed for Quant (Issue #740)

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Hi Fengchao,

I've gone back through my previous run, and adjusting the mio ions to 2 did remove the protein in the MaxLFQ intensity, but not the standard intensity.

Is there a setting that can be altered to have the MaxLFQ intensity values as the output in the reprint.int.tsv, rather than the stendard intensity?

Thanks, Jessica

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