Let IonQuant support the "allow unlabeled"

[helloworld1631]

- Upload your log file
log_2024-05-10_14-52-03-ModTag.txt
log_2024-05-10_15-01-17-none.txt

- Describe the issue or question:
Hello, I found that while the TMT Integrator in my workflow runs successfully, the generated report is empty. Upon reviewing, I discovered that each channel intensity in the psm.tsv file is zero. Interestingly, when I switch the Intensity extraction tool in the Quant(Isobaric) panel from IonQuant to Philosopher, the psm.tsv file then includes the corresponding reporter ion intensities.

However, when proceeding to the TMT Integrator step, it throws an error: 'java.lang.StringIndexOutOfBoundsException: String index out of range: 10.' Through testing, I believe this issue is related to the PTM report; after changing the Mod tag to 'none', it can output the report normally.

My mzml files were generated by MSconvert. Could there be a problem with the parameter settings in my workflow?

Thank you!

[fcyu]

I believe you either uploaded the wrong log file or messed up the setting because both workflow only run TMT-Integrator.

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
CheckCentroid
C:\Users\MSI13900K\Desktop\Fragpipe211\jre\bin\java.exe -Xmx38G -cp C:\Users\MSI13900K\Desktop\Fragpipe211\lib\fragpipe-21.1.jar;C:\Users\MSI13900K\Desktop\Fragpipe211\tools\batmass-io-1.30.0.jar com.dmtavt.fragpipe.util.CheckCentroid C:\Users\MSI13900K\Desktop\NormalSearching\RawFiles\IodoTMT_Ectodomain_20230724\E_ZW_IodoTMT__20230724_F10.mzML 24
Done in 1.0 s.
Process 'CheckCentroid' finished, exit code: 0
TmtIntegrator [Work dir: C:\Users\MSI13900K\Desktop\NormalSearching\IodoTMT_Searching3]
C:\Users\MSI13900K\Desktop\Fragpipe211\jre\bin\java.exe -Xmx38G -cp C:\Users\MSI13900K\Desktop\Fragpipe211\tools\tmt-integrator-5.0.7.jar TMTIntegrator C:\Users\MSI13900K\Desktop\NormalSearching\IodoTMT_Searching3\tmt-integrator-conf.yml C:\Users\MSI13900K\Desktop\NormalSearching\IodoTMT_Searching3\psm.tsv
TMT-Integrator v5.0.7
Load parameters and check PSMs--- 0.00092 min.
UpdateColumns--- 0.00558 min.
LoadPsms--- 0.00033 min.
Take log and normalize--- 0.00043 min.
PSM normalization--- 0.00000 min.
outlierRemoval--- 0.00052 min.
Collapse--- 0.00047 min.
protNorm--- 0.00000 min.
Report--- 0.00017 min.
-----------------------------------------------------------------------
LoadPsms--- 0.00042 min.
Take log and normalize--- 0.00023 min.
PSM normalization--- 0.00000 min.
outlierRemoval--- 0.00043 min.
Collapse--- 0.00017 min.
protNorm--- 0.00002 min.
Report--- 0.00027 min.
-----------------------------------------------------------------------
LoadPsms--- 0.00020 min.
Take log and normalize--- 0.00023 min.
PSM normalization--- 0.00000 min.
outlierRemoval--- 0.00027 min.
Collapse--- 0.00017 min.
protNorm--- 0.00003 min.
Report--- 0.00010 min.
-----------------------------------------------------------------------
LoadPsms--- 0.00020 min.
Take log and normalize--- 0.00022 min.
PSM normalization--- 0.00000 min.
outlierRemoval--- 0.00028 min.
Collapse--- 0.00015 min.
protNorm--- 0.00000 min.
Report--- 0.00010 min.
-----------------------------------------------------------------------
LoadPsms--- 0.00020 min.
Take log and normalize--- 0.00020 min.
PSM normalization--- 0.00000 min.
outlierRemoval--- 0.00028 min.
Collapse--- 0.00015 min.
protNorm--- 0.00002 min.
Report--- 0.00008 min.
-----------------------------------------------------------------------
LoadPsms--- 0.00022 min.
Take log and normalize--- 0.00018 min.
PSM normalization--- 0.00000 min.
outlierRemoval--- 0.00030 min.
Collapse--- 0.00015 min.
protNorm--- 0.00003 min.
Report--- 0.00010 min.
-----------------------------------------------------------------------
LoadPsms--- 0.00022 min.
Take log and normalize--- 0.00020 min.
PSM normalization--- 0.00000 min.
outlierRemoval--- 0.00015 min.
Collapse--- 0.00033 min.
protNorm--- 0.00000 min.
Report--- 0.00023 min.
-----------------------------------------------------------------------
LoadPsms--- 0.00030 min.
Take log and normalize--- 0.00020 min.
PSM normalization--- 0.00000 min.
outlierRemoval--- 0.00018 min.
Collapse--- 0.00038 min.
protNorm--- 0.00010 min.
Report--- 0.00027 min.
-----------------------------------------------------------------------
LoadPsms--- 0.00027 min.
Take log and normalize--- 0.00027 min.
PSM normalization--- 0.00000 min.
outlierRemoval--- 0.00015 min.
Collapse--- 0.00023 min.
protNorm--- 0.00008 min.
Report--- 0.00052 min.
-----------------------------------------------------------------------
Total run time--- 0.01812 min.
Finish!!!
Process 'TmtIntegrator' finished, exit code: 0

Please cite:
(Isobaric-labeling quantification) Quantitative proteomic landscape of metaplastic breast carcinoma pathological subtypes and their relationship to triple-negative tumors. Nat Commun. 11:1723 (2020)
(Visualization with FragPipe-PDV) PDV: an integrative proteomics data viewer. Bioinformatics. 35(7):1249 (2019)

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Best,

Fengchao

[helloworld1631]

Hi Fengchao,

To troubleshoot, I only ran the TMT-Integrator but with different settings. The file "log_2024-05-10_14-52-03-ModTag.txt" contains settings where I added the PTM tag to generate PTM-specific reports. The file "log_2024-05-10_15-01-17-none.txt" did not have the PTM tag set. If necessary, I can also provide the other part of log and the psm.tsv file.

[fcyu]

Yes, please upload the other log file containing the whole process, first.

Thanks.

Fengchao

[helloworld1631]

Hi Fengchao,

Here are the log files
log_2024-05-10_11-59-18_searching_and_validation.txt
log_2024-05-10_13-59-06_extract_with_IonQuant.txt
log_2024-05-10_14-45-47_extract_with_Philospher.txt
log_2024-05-10_14-52-03-ModTag.txt
log_2024-05-10_15-01-17-none.txt
and psm.tsv.
psm.zip

Thank you!

[fcyu]

You set the TMT-6 but the variable modifications does not seems to be TMT-6:

variable_mod_09 = 417.22485 K 2
variable_mod_12 = 417.22485 n^ 1

May I ask what isobaric label you were using?

Thanks,

Fengchao

[helloworld1631]

Hi Fengchao,

This mass shift comes from NHS-ester probe with iodoTMTsixplex. The modification mass is not the common 229 but the reporter ions series should be the same.

Thank you!

[fcyu]

Thanks for the clarification.

The current version of IonQuant filters out the PSMs without any variable modifications corresponding to the isobaric label. That was why using IonQuant as the intensity extraction tool got zero intensities.

I see that you enabled "allow unlabeled" in the Quant (Isobaric) tab. I think I should let IonQuant take this parameter and extract the reporter ion intensities for the cases like yours.

Thanks again for the feedback.

Best,

Fengchao

[helloworld1631]

Hi Fengchao,

Thank you for considering my case for software feature updates. I noticed that you changed the issue title. Shall I create a new issue for the discussion about the PTM-specific report generation error?

Thank you!

[fcyu]

No prob! And no need to create another issue, I will use this one to track the changes.

Best,

Fengchao

[helloworld1631]

I see. What do you think is the reason for the other problem I met, where it fails to generate a PTM-specific report? Because when I checked the psm file, Philosopher had already provided the reporter ion intensity for each channel.

Thank you!

[fcyu]

What do you think is the reason for the other problem I met, where it fails to generate a PTM-specific report?

Because the format was wrong: n(417.2249),K(417.2249)\:probe. It should be n(417.2249),K(417.2249) (not 100% sure that the N-term mod will work though).

Best,

Fengchao