Help with PTM analysis

[Shelly Kalaora]

Hi,

I performed activity-based protein profiling (ABPP), looking at DBIA binding to cysteine sites, in addition to global proteome changes. The samples are multiplexed with TMT-18, and it is a single plex. I analyzed the data using FragPipe, with a workflow similar to their "chemprot-ABPP-IADTB-TMT" (see attached mods) 

I am trying to analyze the data using MSstats shiny, with the following selections:

Biological Question: PTM

Label Type: TMT

Type of File: FragPipe

I uploaded the msstat files for the PTM and proteome, as well as the MSstatsTMT_annotation file.

For the following: "Please enter the name of the modification id column", I tried to enter "C", but it gave the error: "Failed to load data: Multiple columns found containing mod_id_col string. Please be more specific and add full column name (including mass).", so I tried to add the exact mass in the format: "C:239.1634". But it gives the error: "Failed to load data: subscript out of bounds".

Can you please help me understand the problem?

Thank you,

Shelly

[Anthony Wu]

Hi Shelly,

Instead of "C:239.1634", could you try "C.239.1634" (i.e. I'm replacing the colon with a period)?  

I had another Fragpipe dataset and noticed the column names with the PTM sites would be in the format <amino acid>.<mass> instead of <amino acid>:<mass>.  If this is also the case with you, then I'll need to update the tooltip of the "Please enter the name of the modification id column" section in MSstatsShiny since it looks outdated/misleading.

Thanks,

Tony

[Shelly Kalaora]

Hi Tony,

I tried it, but it still gives an error: "Failed to load data: subscript out of bounds".

I attached a shortened version of the msstat files (they are too big to upload here), I added if they are the PTM or protein, if it can help to solve the problem?

Thank you,

Shelly

[Anthony Wu]

Hi Shelly,

Thank you for providing a sample of the dataset.  This is very helpful.

I noticed in the PTM dataset, there is no column named "C.239.1634".  Typically, if there is a PTM in your dataset, we expect a column in the format of <amino acid>.<mass> to exist.  Are you able to generate a dataset from Fragpipe with this column?

Thanks,

Tony

[Shelly Kalaora]

Hi Tony,

This is the output that the FragPipe gave me. I am usually not working with  FragPipe  , but I followed their instructions (it is based on this: https://fragpipe.nesvilab.org/docs/tutorial_abpp.html). I attached the workflow file. Can you help me look for anything that is missing to create the column that is needed?

Thank you,

Shelly

[Anthony Wu]

Hi Shelly,

I'm afraid I might not be the best person to figure out how to add this column as I personally don't use Fragpipe often.  I would recommend asking the developers of Fragpipe how to do this.

Thanks,

Tony