Glycolibrary reuse

[ecamafeita]

Hi, I had Fragpipe successfully create a library.tsv file using the glyco-N-DIA module with DDA data. It also performed quantitation in some DIA raw files in the same run. Since creating the library took quite a long time, I wonder if it's now possible to reuse this library.tsv file to quantitate some additional DIA files. Thank you.

[dpolasky]

Hi,
Yes you can. Load the new DIA files on the workflow tab, select the library.tsv file from the first run as the spectral library file on the Quant (DIA) tab, and turn off all tools prior to DIA quant (uncheck 'run MSFragger', 'run validation tools', etc. on each tab before Quant (DIA)).

Best,
Dan

[ecamafeita]

Thank you, Dan... so I did, except for the "Database" tab, where a database had to be necessarily specified. But I got error messages related to FragPipe's attempts to read uncalibrated.mzML files that had not been previously created (I used RAW files as inputs):
ERROR: Failure reading input file S:\DIARawFiles\Peptides_Box1_4_uncalibrated.mzML.
I'm using version 23.1, don't if this matters.

   Cheers,

   Emilio.   

[fcyu]

@ecamafeita you will need to convert your DIA quant file to mzML format following the tutorial here https://fragpipe.nesvilab.org/docs/tutorial_convert.html

Best,

Fengchao

[ecamafeita]

Thanks for your prompt answer, Fengchao.
I used mzML files, then I got this error message: Exception in thread "main" java.lang.RuntimeException: java.io.FileNotFoundException: S:\GlycoDIA_10sample_test\psm.tsv (The system cannot find the file specified).
Seems that FragPipe's looking for the psm.tsv file that should have been generated by the Spec Lib module, but I was told to have this one turned off...

[fcyu]

Could you share the whole log file?

Thanks,

Fengchao

[ecamafeita]

Sure, here it is.

log_2026-02-03_18-03-14.txt

[fcyu]

Could you upgrade FragPipe to 24.0, set the data type to DIA-Quant (not DIA), and try again? I think we have made some changes to the requirement of the psm.tsv file, but I don't remember if that fixed the error you were having.

Thanks,

Fengchao

[ecamafeita]

In doing so, I got an error message at the cross-run analysis stage.
I'm attaching the log file.

     Thank you.

log_2026-02-04_18-41-43.txt

[fcyu]

First of all, you are using a library different from the previous test: 132PlasmaSamplesGlycolibrary.tsv vs library.tsv.

Second, you will need to "declare" the modifications in the "Cmd line opts" textbox of the "Quant (DIA)" tab: --mod [name],[mass],[optional: 'label'] declares a modification name. Examples: "--mod UniMod:5,43.005814", "--mod SILAC-Lys8,8.014199,label".

Last, we have fixed the error that happened in your previous test. If you want to test the fixed version, please unzip and put the attached jar file in the "tools" folder of the FragPipe installation folder, delete the old FragReporter jar file, and restart FragPipe.

FragReporter-1.1.4.jar.zip

Best,

Fengchao

[ecamafeita]

Thank you. The library is the same, it was just renamed and moved to another folder. Regarding the declaration of modifications, this should probably include whether they are variable or fixed modifications. How is that done?

Cheers,

Emilio.

[fcyu]

The library is the same, it was just renamed and moved to another folder.

I doubt that. They have different file names, not just different folders. Also, with your first library, there is no such error.

Regarding the declaration of modifications, this should probably include whether they are variable or fixed modifications. How is that done?

You will need to "declare" the modifications in the "Cmd line opts" textbox of the "Quant (DIA)" tab: --mod [name],[mass],[optional: 'label'] declares a modification name. Examples: "--mod UniMod:5,43.005814", "--mod SILAC-Lys8,8.014199,label".

Best,

Fengchao

[ecamafeita]

The library remains unchanged; only the file name and its location were modified. The error occurred when we moved from version 23.1 to version 24.0.
So I don't need to specify if the modifications are fixed or variable?

   Best,

   Emilio.

[fcyu]

So I don't need to specify if the modifications are fixed or variable?

You need to specify ALL modifications.

Best,

Fengchao