DDA/DIA Hybrid analysis file handling

[WonnShim]

Hi,
Thank you for the wonderful work.

I am new in the field of proteomics, and I am trying to analyze my DDA and DIA data (LFQ) generated by Thermo's orbitrap.
I have couple of questions using Fragpipe in Linux.

1-1. Can I always directly load .raw file instead of .mzML file if I have installed Mono?
1-2. If I have to load .mzML file, which .mzML do I have to load? - calibrated or uncalibrated?

2-1. When analyzing DDA and DIA with hybrid method, I loaded both DDA and DIA files and used 'DIA_SpecLib_Quant' workflow without changing any parameters. Am I doing right?
2-2. When analyzing with hybrid method, do I have to load spectral library generated by DDA in the validation tab manually before analyzing?

3. In MSFragger tab, is setting enzyme to 'stricttrypsin' better than 'trypsin' if I digested my samples with commonly-used trypsin?

4-1. When performing differential expression analysis, should I use 'report.pg_matrix.tsv' file for the input of protein intensity-requiring tools such as Perseus?
4-2. I get a bit different results between Perseus (accepts protein intensity values) vs MSstats(accepts peptide intensity values). How is protein intensity in report.pg_matrix.tsv file calculated from the peptide intensity? (Maybe the different results comes from using different methods between those tools - normalization, imputation, etc...)

Thank you so much for helping me out.

[fcyu]

1-1. Can I always directly load .raw file instead of .mzML file if I have installed Mono?

Yes.

1-2. If I have to load .mzML file, which .mzML do I have to load? - calibrated or uncalibrated?

Calibrated or uncalibrated mzML files are not meant to be used as input files. They are intermediate files in the workflow. If you want to load the mzML file, please use the one converted by MSConvert.

2-1. When analyzing DDA and DIA with hybrid method, I loaded both DDA and DIA files and used 'DIA_SpecLib_Quant' workflow without changing any parameters. Am I doing right?

You need to specify the data types for your input files in the "workflow" tab. You also need to specify the fasta file in the "database" tab.

2-2. When analyzing with hybrid method, do I have to load spectral library generated by DDA in the validation tab manually before analyzing?

No.

3. In MSFragger tab, is setting enzyme to 'stricttrypsin' better than 'trypsin' if I digested my samples with commonly-used trypsin?

Yes.

4-1. When performing differential expression analysis, should I use 'report.pg_matrix.tsv' file for the input of protein intensity-requiring tools such as Perseus?

Yes. You can use our FragPipe-Analyst (http://fragpipe-analyst.nesvilab.org/), which supports the FragPipe output file better, to perform the differential expression analysis.

4-2. I get a bit different results between Perseus (accepts protein intensity values) vs MSstats(accepts peptide intensity values). How is protein intensity in report.pg_matrix.tsv file calculated from the peptide intensity? (Maybe the different results comes from using different methods between those tools - normalization, imputation, etc...)

As far as I know, it is calculated using the MaxLFQ.

Best,

Fengchao

[WonnShim]

Thanks for the prompt response.

I tried using FragPipe-Analyst as you suggested, and it ran well in DIA (protein) mode.
However, I cannot run DIA (peptide) since I can't find 'peptide-level report ( abundance_peptide_[MS1/MS2quant]_[normalization].tsv ) generated by by FragPipe (we recommend abundance_peptide_MS2quant_Norm.tsv file)'-the one that is needed to be loaded- in my hybrid analysis report.

How can I generate that peptide file?

Thanks.

[fcyu]

Thanks for the test and feedback. There is a bug that FragPipe doesn't generate the DIA peptide-level report. It has been fixed. If you want to try the pre-released version, please send an email to yufe AT umich.edu

Thanks,

Fengchao