Issue with TMT Reporter Ion Extraction in IonQuant

[lfu46]

Dear IonQuant Team,

I am encountering an issue with IonQuant related to the extraction of TMT reporter ion intensities in the PSM result table. Due to the specific requirements of my experimental design, I rely on TMT reporter ion intensities in the PSM table for quantification. However, I noticed that many PSMs show a value of 0 for all TMT channels, as illustrated in the attached figure.

Interestingly, these PSMs are identified as TMT-labeled, and upon reviewing the spectra in the raw files, I can confirm the presence of TMT reporter ions (see the example in the figure below).

I am wondering if there might be factors preventing IonQuant from extracting the TMT reporter ions from the spectra. My experiment includes some unique settings: there is a modification on the N-terminus, where the TMT reporter ion is labeled. Consequently, the final N-terminal modification is the adduct of the initial modification and the TMT reagent.

In the results, only PSMs with lysine (K) in their sequences are quantified, with non-zero TMT reporter ion intensities in the PSM table. In contrast, sequences with arginine (R) at the C-terminus or without any lysine show 0 reporter ion intensities. Despite this, the spectra in the raw files indicate the presence of TMT reporter ions even for peptides without lysine. I suspect this may be due to the lower relative abundance of these ions compared to those from peptides with lysine, leading IonQuant to exclude them as low-quality data.

Would it be possible to adjust the settings in MSFragger or IonQuant to ensure the extraction of these reporter ions rather than assigning them a value of 0?

I greatly appreciate your assistance and look forward to your guidance on this matter.

Thank you very much.

Best,
Longping

[anesvi]

FragPipe tools would need to be modified to recognize those masses as TMT labels. As this requires work and is unique to your experiment, we normally do such things as a collaboration. Perhaps you can contact me by email to discuss.
Best,
Alexey

[lfu46]

Hi Prof. Nesvizhskii,

Thank you very much for your kind offer to collaborate—I truly appreciate it.

I just now attempted to use Philosopher to extract and quantify the PSMs, and it seems to have worked successfully. Please find the results in the attached figure:

I was wondering if the differences could be due to the distinct extraction methods employed by IonQuant and Philosopher. Could you kindly explain the differences between these two quantification methods?

Thank you again for your help.

Best,
Longping

[anesvi]

I am not sure, maybe Philosopher extracts reporter ions regardless of whether the PSM is marked as TMT labelled. But I do not think TMT-Integrator will work. But if you only need PSM.tsv files,then it may be sufficient for you to just run Philosopher.

[lfu46]

Thank you for your explanation. I will reach out to you if I encounter any further issues or identify opportunities for collaboration.

Best,
Longping

[fcyu]

That's correct. IonQuant won't extract the channel intensity if there are no TMT labels.

Best,

Fengchao

[lfu46]

Thanks for your help, Fengchao.

Best,
Longping