DIA_SpecLib_Quant workflow exit code: -1073741819

[valersch13]

Hello,

I am new to DIA workflows and tried running the DIA_SpecLib_Quant workflow in Fragpipe v.24 with spectral library generation, without using any additional spectral libraries (see also attached log file).
Unfortunately I got an error code -1073741819 and I am not sure what it means and what I did wrong. Could you please assist me?
Thank you so much!

Best,
Valerie

log_2026-01-22_10-57-33.txt

[fcyu]

The "MBR" checkbox in the "Quant (DIA)" tab should not be enabled, which may be the cause of the error. Could you disable it and try again?

Thanks,

Fengchao

[valersch13]

Thank you that worked!
Could you please also tell me if this version of Fragpipe supports generating spectral libraries for DIA-MS with DDA raw files? And how would this be done correctly since I can't find a tsv file in normal LFQ-MBR results that could be used as a spectral library for DIA or am I mistaken?

[fcyu]

Yes, just use the same DIA_SpecLib_Quant workflow, load your DDA files, and specify them as the "DDA" data type.

Best,

Fengchao

[valersch13]

Great, thank you! Can I disable the spectral library generation, when running DIA with the DDA spectral library in the DIA_SpecLib_Quant workflow or does that mess up analysis/algorithm?

[fcyu]

Maybe I misunderstand what you want to do. Do you want to use your DDA runs to build a spectral library and then do the DIA quant using that library? Or do you already have the library?

Thanks,

Fengchao

[valersch13]

Yes, I have several trail runs and one of them is to use the DDA runs to build a spectral library and then do the DIA quant using that library.

[fcyu]

OK, then, my understanding is correct. You just need to load both DDA and DIA runs, and specify them as "DDA" and "DIA" types, respectively. Load the DIA_SpecLib_Quant workflow and do not disable the spectral libray building.

Best,

Fengchao

[valersch13]

Just to avoid misunderstanding, you mean upload the DDA and DIA raw files together, specify them accordingly if not automatically recognised as DDA and DIA files accordingly and just run the DIA_SpecLib_Quant with spectral library building enabled?

[fcyu]

Yes.

[valersch13]

Does that mean the spectral library is build from DIA and DDA files together then? If I only want my DDA data to be the spectral library for the DIA data quantification, can I then only upload my DIA files, disable spectral library building and upload the spectral library from the previous DDA run for quantification or is quantification like that not entirely supported with the workflows in FragPipe?

[fcyu]

Oh, if you want the library only from the DDA data, set the DIA run to the “DIA-Quant” data type in the process I described.

Best,

Fengchao

[valersch13]

Great, thank you so much!