Closed bynjh007 closed 1 month ago
Hi Jin,
iTRAQ should work, but you will have to manually list the reporter ions, like below. Also note that a tmt.tsv
file will still be written, and the channel names will be tmt_1
, tmt_2
, ...
Please report any issues you find!
"quant": {
"tmt": {
"User": [
114,
115,
116,
117
]
},
"tmt_settings": {
"level": 3,
"sn": false
}
}
Thanks for your guidance! I have one more question. Is there a parameter for mass tolerance for ITRAQ (or TMT) reporter ions?
At the moment it is hard-coded to 20 ppm. I am open to reifying it as a full user-modified parameter if needed!
At the moment it is hard-coded to 20 ppm. I am open to reifying it as a full user-modified parameter if needed! I think this would be a very useful option!
According to your help, now I generated a json file for testing one of my data (MS-spec: Q Exactive Orbitrap).
But I keep facing runtime error:
thread '
I noticed that "max_len 20" works, but "max_len 30" causes that error. But I need at least 40. Would you recommend how this can be solved?
Thanks!
json file: { "database": { "bucket_size": 16384, "enzyme": { "missed_cleavages": 2, "min_len": 7, "max_len": 50, "cleave_at": "KR", "restrict": "P", "c_terminal": true, "semi_enzymatic": true }, "fragment_min_mz": 150.0, "fragment_max_mz": 1500.0, "peptide_min_mass": 500.0, "peptide_max_mass": 5000.0, "ion_kinds": [ "b", "y" ], "min_ion_index": 2, "max_variable_mods": 2, "static_mods": { "C": 57.0214, "^": 144.1021, "K": 144.1021 }, "variable_mods": { "M": 15.9949, "N": 0.9840, "Q": 0.9840 }, "decoytag": "rev", "generate_decoys": true, "fasta": "/home/reference/uniprot/human_contam_20240723.fasta" }, "precursor_tol": { "ppm": [-10,10] }, "fragment_tol": { "ppm": [-10,10] }, "precursor_charge": [2, 4], "isotope_errors": [0,0], "wide_window": false, "chimera": false, "report_psms": 5,
"deisotope": true,
"min_peaks": 15,
"max_peaks": 150,
"min_matched_peaks": 4,
"max_fragment_charge": 1,
"quant": {
"tmt": {
"User": [114,115,116,117]
},
"tmt_settings": {
"level": 2,
"sn": false
}
},
"mzml_paths": [
"/home/bynjh007/eogc/global/N111T112.mzML",
]
}
Your configuration will likely require 1+ TB of RAM to successfully complete. I strongly recommend the following alterations:
Your configuration will likely require 1+ TB of RAM to successfully complete. I strongly recommend the following alterations:
- Do not use semi-enzymatic search
- Remove N/Q deamidation variable modifications
- Widen your precursor tolerance to "da": [-3.5, 1.25]. This will encompass isotope errors (which you are not using) and deamidation.
Thanks for the suggestion. turning off the "semi-enzymatic search" mode only make it work!
However, in my tmt.tsv, most of values in tmt1_4 are 0 (also ion_injection_time=0), which means that it didn't properly capture iTRAQ reporter ion.(114, 115, 116, 117).I also changed fragment_min_mz to 100, but it didn't solve the problem.
Could you help me how I can solve this and obtain the proper iTRAQ values?
Thanks!
If you're using the most recent release, fragment_min_mz should be automatically accounted for when using MS2-based reporter ion quant. The most likely oversight is trimming out the reporter ion peaks if they aren't in the top 150 by intensity. Can you try setting the max_peaks
parameter to something like 500 or 1000 and see if that resolves the issue?
If you're using the most recent release, fragment_min_mz should be automatically accounted for when using MS2-based reporter ion quant. The most likely oversight is trimming out the reporter ion peaks if they aren't in the top 150 by intensity. Can you try setting the
max_peaks
parameter to something like 500 or 1000 and see if that resolves the issue?
Yes, I am using the v0.14.7 (from bioconda). I increased max_peaks to 1000, and the number of non-zero itraq values are slightly increased but still, majorities are zero. Also, non-zero itraq values were only detected in one of the four reporter ions (either 114 or 115 or 116 or 117) like this.
Here are my json: { "database": { "bucket_size": 8192, "enzyme": { "missed_cleavages": 2, "min_len": 7, "max_len": 50, "cleave_at": "KR", "restrict": "P", "c_terminal": true, "semi_enzymatic": false }, "fragment_min_mz": 100.0, "fragment_max_mz": 1500.0, "peptide_min_mass": 500.0, "peptide_max_mass": 5000.0, "ion_kinds": [ "b", "y" ], "min_ion_index": 2, "max_variable_mods": 2, "static_mods": { "C": 57.0214, "^": 144.1021, "K": 144.1021 }, "variable_mods": { "M": 15.9949, "N": 0.9840, "Q": 0.9840 }, "decoytag": "rev", "generate_decoys": true, "fasta": "/home/reference/uniprot/human_contam_20240723.fasta" }, "precursor_tol": { "ppm": [-10,10] }, "fragment_tol": { "ppm": [-10,10] }, "precursor_charge": [2, 4], "isotope_errors": [0,0], "wide_window": false, "chimera": false, "report_psms": 5,
"deisotope": true,
"min_peaks": 15,
"max_peaks": 1000,
"min_matched_peaks": 4,
"max_fragment_charge": 1,
"quant": {
"tmt": {
"User": [114,115,116,117]
},
"tmt_settings": {
"level": 2,
"sn": false
}
},
"mzml_paths": [
"/home/bynjh007/eogc/global/N111T112.mzML"
]
}
Ah, you'll need to actually find and use the real (e.g. high resolution) iTRAQ reporter ion m/z values - I just put dummy ones in there :)
You mean [114, 115, 116, 117] in quant:tmt:user? I assumed that iTRAQ 4-plex reporter ion m/z is also 114-117?
Yes, fill in the reporter ion mz values with more significant figures. E.g. For TMT126 you would use 126.127726, not just 126
Thanks for this powerful tool,
Is this tool also available for iTRAQ labeling data? I am planning to use this tool to reanalyse my previous data (iTRAQ-global, phospho, glycoproteome data).
Best, Jin