The same protein group has different protein IDs in protein.tsv files from different experiments

[jd690764]

• Describe the issue or question: Hi,

I am trying to quantitate a group of timsTof dda experiments using ionQuant and close to end of the process I got an error that I don't quite understand. Could you help me find out what the issue is and how to get around it?

Thank you, Janos

• Upload your log file (If a log file hasn't been generated, go to the 'Run' tab in FragPipe, click 'Export Log', zip the resulting "log_[date_time].txt" file to avoid truncation, then attach the zipped file by drag & drop here. log_2021-11-09_20-49-57_error.txt )

[fcyu]

Hi Janos,

The error message is The protein group 3053 and subgroup a in G:\Mohammad_2103_\210330_nasal_drugs\nasal_imac_msf2\CAY_1/protein.tsv are not consistent with those in another protein.tsv, which means that the protein group 3053 a has different protein IDs in you protein.tsv. It should not happen. Can you help to check? You can also send all of your protein.tsv to us to take a look.

Thanks,

Fengchao

[jd690764]

Hi Fengchao,

This is very strange, here is the protein that is causing the trouble:

group sub_group      protein   protein_id   entry_name gene length
               ds

1: 984 a NP_001349898 NP_001349898 NP_001349898 795 ./CAY_1/protein.tsv 2: 3053 a NP_001349898 NP_001349898 NP_001349898 364 ./CAY_1/protein.tsv 3: 3053 a NP_001349898 NP_001349898 NP_001349898 364 ./CAY_2/protein.tsv 4: 984 a NP_001349898 NP_001349898 NP_001349898 795 ./DMSO_1/protein.tsv 5: 3053 a NP_001349898 NP_001349898 NP_001349898 364 ./DMSO_1/protein.tsv 6: 3053 a NP_001349898 NP_001349898 NP_001349898 364 ./DMSO_2/protein.tsv 7: 3053 a NP_001349898 NP_001349898 NP_001349898 364 ./DMSO_3/protein.tsv 8: 3053 a NP_001349898 NP_001349898 NP_001349898 364 ./Forskolin_1/protein.tsv 9: 3053 a NP_001349898 NP_001349898 NP_001349898 364 ./Forskolin_2/protein.tsv 10: 3053 a NP_001349898 NP_001349898 NP_001349898 364 ./Forskolin_3/protein.tsv 11: 3053 a NP_001349898 NP_001349898 NP_001349898 364 ./SPIKE_1/protein.tsv 12: 3053 a NP_001349898 NP_001349898 NP_001349898 364 ./SPIKE_2/protein.tsv 13: 3053 a NP_001349898 NP_001349898 NP_001349898 364 ./SPIKE_3/protein.tsv

I double checked it, it's only present once in the library. (A side question: What does subgroup mean here?)

Thank you, Janos

On Wed, Nov 10, 2021 at 6:24 AM Fengchao @.***> wrote:

Hi Janos,

The error message is The protein group 3053 and subgroup a in G:\Mohammad_2103_\210330_nasal_drugs\nasal_imac_msf2\CAY_1/protein.tsv are not consistent with those in another protein.tsv, which means that the protein group 3053 a has different protein IDs in you protein.tsv. It should not happen. Can you help to check? You can also send all of your protein.tsv to us to take a look.

Thanks,

Fengchao

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[fcyu]

Hi Janos,

Thanks for your info. Can you show me all proteins with 3053 a and 984 a groups?

Thanks,

Fengchao

[jd690764]

Here they are:

group sub_group      protein   protein_id   entry_name gene length
                            protein_description

ds 1: 984 a NP_001349898 NP_001349898 NP_001349898 795 zinc finger C2HC domain-containing protein 1A isoform 2 ./CAY_1/protein.tsv 2: 3053 a NP_001349898 NP_001349898 NP_001349898 364 zinc finger C2HC domain-containing protein 1A isoform 2 ./CAY_1/protein.tsv 3: 984 a NP_001349997 NP_001349997 NP_001349997 795 cell cycle and apoptosis regulator protein 2 isoform 2 ./CAY_2/protein.tsv 4: 3053 a NP_001349898 NP_001349898 NP_001349898 364 zinc finger C2HC domain-containing protein 1A isoform 2 ./CAY_2/protein.tsv 5: 984 a NP_001349898 NP_001349898 NP_001349898 795 zinc finger C2HC domain-containing protein 1A isoform 2 ./DMSO_1/protein.tsv 6: 3053 a NP_001349898 NP_001349898 NP_001349898 364 zinc finger C2HC domain-containing protein 1A isoform 2 ./DMSO_1/protein.tsv 7: 3053 a NP_001349898 NP_001349898 NP_001349898 364 zinc finger C2HC domain-containing protein 1A isoform 2 ./DMSO_2/protein.tsv 8: 3053 a NP_001349898 NP_001349898 NP_001349898 364 zinc finger C2HC domain-containing protein 1A isoform 2 ./DMSO_3/protein.tsv 9: 984 a NP_001349989 NP_001349989 NP_001349989 795 microtubule-associated tumor suppressor 1 isoform 9 ./Forskolin_1/protein.tsv 10: 3053 a NP_001349898 NP_001349898 NP_001349898 364 zinc finger C2HC domain-containing protein 1A isoform 2 ./Forskolin_1/protein.tsv 11: 3053 a NP_001349898 NP_001349898 NP_001349898 364 zinc finger C2HC domain-containing protein 1A isoform 2 ./Forskolin_2/protein.tsv 12: 3053 a NP_001349898 NP_001349898 NP_001349898 364 zinc finger C2HC domain-containing protein 1A isoform 2 ./Forskolin_3/protein.tsv 13: 3053 a NP_001349898 NP_001349898 NP_001349898 364 zinc finger C2HC domain-containing protein 1A isoform 2 ./SPIKE_1/protein.tsv 14: 3053 a NP_001349898 NP_001349898 NP_001349898 364 zinc finger C2HC domain-containing protein 1A isoform 2 ./SPIKE_2/protein.tsv 15: 3053 a NP_001349898 NP_001349898 NP_001349898 364 zinc finger C2HC domain-containing protein 1A isoform 2 ./SPIKE_3/protein.tsv

Thanks, Janos

On Wed, Nov 10, 2021 at 9:08 AM Fengchao @.***> wrote:

Hi Janos,

Thanks for your info. Can you show me all proteins with 3053 a and 984 a groups?

Thanks,

Fengchao

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[fcyu]

Thanks Janos,

This is a bug in Philosopher that creates the same protein group ID with different proteins. For group 984 a, there are protein.tsv having protein NP_001349898, NP_001349997, and NP_001349989. Theoretically, the same protein group always has the same protein ID.

Hopefully, Felipe @prvst can fix this bug soon.

Best,

Fengchao

[prvst]

Hi @fcyu, philosopher does not create or alter the protein groups, it borrows from ProteinProphet, which is the program that makes them. I need to see more data before guessing what's the issue. @jd690764, could you send me your prot.xml, psm.tsv, and protein.tsv files?

[jd690764]

Hi Felipe,

I gave you access to the data with your umich email - I sent you an email to that address. Please, let me know if there are any issues accessing it.

Thank you, Janos

On Wed, Nov 10, 2021 at 10:05 AM Felipe da Veiga Leprevost < @.***> wrote:

Hi @fcyu https://github.com/fcyu, philosopher does not create or alter the protein groups, it borrows from ProteinProphet, which is the program that makes them. I need to see more data before guessing what's the issue. @jd690764 https://github.com/jd690764, could you send me your prot.xml, psm.tsv, and protein.tsv files?

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[prvst]

@jd690764 The file share is requesting an authentication from my @med.umich account, which does not exist (in Google). To make it easier, I'm sending you a file share request via my Dropbox

[jd690764]

Hi Felipe,

I uploaded the data - thank you for looking into this.

Janos

On Fri, Nov 12, 2021 at 7:40 AM Felipe da Veiga Leprevost < @.***> wrote:

@jd690764 https://github.com/jd690764 The file share is requesting an authentication from my @med.umich account, which does not exist (in Google). To make it easier, I'm sending you a file share request via my Dropbox https://www.dropbox.com/request/nxNI1LOApZ8Ax3k3ob7k

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[Monod79]

Hi everyone,

Maybe this can help, I've got the same issue as Janos while running FragPipe v17.0. I ran the same analysis with FragPipe v16.1 build 5 and everything ran smoothly with no error.

Joe

[fcyu]

Thanks Joe,

Can you send your files to us? I can give you a link to upload if you need one.

Thanks,

Fengchao

[Monod79]

Of course !

[fcyu]

Thank you! Here is the link: https://www.dropbox.com/request/kG1NENHn00REagbkFepR

Best,

Fengchao

[Monod79]

Which files do you need Fengchao?

[fcyu]

Spectral files (mzML, raw, or .d), fragger.params, fasta file, and the log file.

Thanks,

Fengchao

[Monod79]

Files have been uploaded.

Thanks for your help Fengchao !

[prvst]

@jd690764 can you also upload your database?

[jd690764]

Hi Felipe,

I uploaded the library.

Thanks, Janos

On Tue, Nov 16, 2021 at 6:25 AM Felipe da Veiga Leprevost < @.***> wrote:

@jd690764 https://github.com/jd690764 can you also upload your database?

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[prvst]

I found and corrected a mismatch between ProteinProphet, and Philosopher. Its seems to have resolved both cases. The fix will be added to the next release. Thanks for the feedback.

[jd690764]

Thank you, Felipe!

When do you think the new update will be available?

Janos

On Tue, Nov 16, 2021 at 10:18 AM Felipe da Veiga Leprevost < @.***> wrote:

I found and corrected a mismatch between ProteinProphet, and Philosopher. Its seems to have resolved both cases. The fix will be added to the next release. Thanks for the feedback.

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[Monod79]

Thanks Felipe!

[jd690764]

Hi Felipe,

v17.1 is working fine!

Thanks, Janos

On Tue, Nov 16, 2021 at 11:54 AM Jonathan Boulais @.***> wrote:

Thanks Felipe!

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[fcyu]

Hi Felipe @prvst ,

Have you made any changed in Philosopher for this issue? If not, I don't think this issue has been fixed. There are two users in this thread. I have @Monod79 's file and reproduced the issue. I am putting my files to Z:\yufe\for_Felipe\github_fragpipe_523.

Best,

Fengchao