0% Validated Coverage and 0 Validated Peptides in PeptideShaker Reports 6 & 9

[umutcakir]

Dear Team,

First of all, thank you for developing such a great tool.

I have a question regarding the Default Peptide and Protein Reports (Reports 6 and 9 in ReportCLI). I ran SearchGUI using the X!Tandem, OMSSA, and Comet search engines, and then processed the results with PeptideShaker. While running PeptideShaker, I requested the reports using the -reports "0,6,9" option.

In the generated reports, I noticed some unexpected results (at least it is unexpected for me). Specifically, there are no validated peptides or PSMs reported for some proteins in the Protein Report. The validated coverage is shown as 0%, both validated and unique peptides are listed as zero, and Spectrum Counting is also zero. For reference, I have included a portion of the results in the table below. My question is: if there are no validated peptides or PSMs, how are these proteins shown in the Default Reports? Is this expected behavior, or might there be a validation setting I have missed?

Protein Inference	Single Protein	Single Protein	Single Protein
Validated Coverage [%]	0	0	0
All Coverage [%]	64.1	62.07	46.67
Possible Coverage [%]	82.05	82.76	86.67
#Peptides	14	8	2
#Validated Peptides	0	0	0
#Unique Peptides	14	8	2
#Validated Unique Peptides	0	0	0
#PSMs	41	12	5
#Validated PSMs	0	0	0
Confidently Localized Modification Sites
#Confidently Localized Modification Sites
Ambiguously Localized Modification Sites
#Ambiguously Localized Modification Sites
Spectrum Counting	0	0	0
MW [kDa]	4.565371	3.348712	3.515998
Confidence [%]	96.33508	94.7644	92.07547
Validation	Doubtful	Doubtful	Doubtful

Thank you very much for your time.

[hbarsnes]

That indeed looks a bit strange. Have you had a closer look at some of these proteins to see of the results match what you see when looking at the same proteins in PeptideShaker?

Would it be possible for you to share the PeptideShaker project? If so, I can always take a closer look myself?

And sorry for the very late reply.

[umutcakir]

Thank you for your reply. The PeptideShaker files can be accessed via the following link: https://owncloud.gwdg.de/index.php/s/XJQSmSYE6DTqETo

Thank you very much for your time and consideration.

[hbarsnes]

I'm having some trouble unzipping the shared files. Perhaps it is easier if you use the export option in PeptideShaker directly? After opening the project you'll find it via "Export > PeptideShaker Project As > Zip File" in the menu at the top left.

[umutcakir]

Dear Harald,

Thank you very much for your time and support. I have uploaded the file at the link below:
https://drive.google.com/drive/folders/1QnaTAY5Q1NeIYmkgBLlZhObtv_cKdpb_?usp=sharing

The file was generated using PeptideShakerCLI, and I hope it can be opened without any issues.

[hbarsnes]

Thanks for sharing the data! I can confirm that I'm able to open the project in PeptideShaker. Can you provide the accession number for one of the proteins that end up in the report despite having no validated peptides or PSMs?

[umutcakir]

Thank you for your reply. These are proteins that have 0% Validated Coverage and 0 Validated Peptides: MtrunA17_Chr5g0436381_1R_1890-1979_90
MtrunA17_Chr8g0347491_3R_254-376_123
MtrunA17_Chr4g0002311_1R_239-334_96
MtrunA17_Chr2g0294991_3R_123-203_81
MtrunA17_Chr8g0357041_3R_306-428_123
MtrunA17_Chr6g0449151_2R_3-146_144
MtrunA17_Chr1g0188231_2R_549-1001_453