How to create a custom TMT set?

[witszymanski]

Hi all!

Is there a way to create a custom set of TMT tags? In general what we did, is to take the 16plex and divide it into two sets of 9-plex that we can use. I used one of it in my experiment. The experimental design looks like this:

	tag	Mix 1	Mix 2
1	TMTpro16plex-Lys127C	cond1_rep1	cond1_rep3
2	TMTpro16plex-Lys128C	cond1_rep2	cond1_rep4
3	TMTpro16plex-Lys129C	cond2_rep1	cond2_rep1
4	TMTpro16plex-Lys130C	cond2_rep2	cond2_rep2
5	TMTpro16plex-Lys131C	cond3_rep1	cond3_rep1
6	TMTpro16plex-Lys132C	cond3_rep2	cond3_rep2
7	TMTpro16plex-Lys133C	cond4_rep1	cond4_rep1
8	TMTpro18plex-Lys134C	cond4_rep2	cond4_rep2
9	TMTpro18plex-Lys135N	reference	reference

So I will have 2 samples after mixing. Then what I do is:

So I will have full proteome samples for normalization of the protein level and phos-enriched samples for the phosphorylation level.

I am doing the TMT analysis with FragPipe for the first time and I am lost completely. I was trying to follow the tutorial, but I don't know how to make the custom TMT tags. Any help or pointing a direction where I can get one, would be much appreciated!

Greets Witek

[fcyu]

Hi Witek,

What is the "custom TMT tags"? With TMT 16, you can specify sample names for 9 of them and set NA for rest of 7 when editing the annotation table in the Quant (Isobaric) tab:

For different mix, you can assign them to different experiments in the workflow tab.

Best,

Fengchao

[anesvi]

Just to add a small correction, you will want to load TMT16 workflow, but in Isobaric Quant tab change to TMT-18 and then annotate channels to samples Y ou also need to use unique sample names across all plexes (including the reference). For example, unless you have a typo, and you really using the same sample, then add some unique tag, like mix1

3 TMTpro16plex-Lys129C cond2_rep1-mix1 cond2_rep1-mix2 Or, as I suspect you made cut and paste typos, correct as follows:

tag Mix 1 Mix 2 1 TMTpro16plex-Lys127C cond1_rep1 cond1_rep3 2 TMTpro16plex-Lys128C cond1_rep2 cond1_rep4 3 TMTpro16plex-Lys129C cond2_rep1 cond2_rep3 4 TMTpro16plex-Lys130C cond2_rep2 cond2_rep4 5 TMTpro16plex-Lys131C cond3_rep1 cond3_rep3 6 TMTpro16plex-Lys132C cond3_rep2 cond3_rep4 7 TMTpro16plex-Lys133C cond4_rep1 cond4_rep3 8 TMTpro18plex-Lys134C cond4_rep2 cond4_rep4 9 TMTpro18plex-Lys135N Reference1 Reference2

Also add a number to 'Reference', see above. In Isobaric quant tab, set reference tag to 'Reference', so the program will find the reference tag in each sample.

As Fengchao said, all unused TMT channels should be set to NA as sample name

Alexey

From: Fengchao @.> Sent: Friday, November 3, 2023 9:11 AM To: Nesvilab/FragPipe @.> Cc: Subscribed @.***> Subject: Re: [Nesvilab/FragPipe] How to create a custom TMT set? (Issue #1318)

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Hi Witek,

What is the "custom TMT tags"? With TMT 16, you can specify sample names for 9 of them and set NA for rest of 7 when editing the annotation table in the Quant (Isobaric) tab: [image]https://user-images.githubusercontent.com/6926299/280287684-7926e812-9f70-44c0-9b3a-f466d8d3984b.png

[image]https://user-images.githubusercontent.com/6926299/280287489-44cee5a4-2c30-4dfd-80e2-1f1f2269407c.png

For different mix, you can assign them to different experiments in the workflow tab.

Best,

Fengchao

Reply to this email directly, view it on GitHubhttps://github.com/Nesvilab/FragPipe/issues/1318#issuecomment-1792412967, or unsubscribehttps://github.com/notifications/unsubscribe-auth/AIIMM6YFQUQS6FK42UII7ADYCTUN7AVCNFSM6AAAAAA64HFIJ2VHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMYTOOJSGQYTEOJWG4. You are receiving this because you are subscribed to this thread.Message ID: @.@.>>

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[witszymanski]

Hi @anesvi and @fcyu !

I can't stress out how awesome it is to have such a great support here from your side! I set up everything in the way you described, and I think it was all completely correct... Up to the point when I realized, that the Bruker .d folders are not yet supported. I again forgot about that.

Next time, I have some TMT measured with Orbitrap, I'll check it out, or we'll wait for some FragPipe update :)

Thanks again and sorry to bother you in the lost case from the get go.

Greets Witek

[witszymanski]

Will it work with TT-TMT data if I cinvert them to mzML?

LIke with this: https://github.com/gtluu/timsconvert https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9991885/

[fcyu]

Unfortunately, no. The incompatibility is not due to the file format but the additional ion mobility dimension. Converting it to mzML doesn't help. We will add the support and let you know when it is ready.

Best,

Fengchao