anesvi
commented
2 years ago I think Dan wrote a tutorial on that part. If not, Dan – we should put something together, see below
From: MNTsnowman @.> Sent: Saturday, November 13, 2021 2:20 PM To: Nesvilab/FragPipe @.> Cc: Subscribed @.***> Subject: [Nesvilab/FragPipe] Quesions for glycan workflows (Issue #526)
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Hi Fragpipe
I’m unsure of where to ask this question, so I’ll drop it here… If you prefer to take it by mail or something else, let me know and we can do that. I have been wanting to explore the glycoproteome of my samples, but I have no clue how to do it effectively and how to interpret the output. In Fragpipe V 17 I noted that the PTM tab is changed quite a lot with stuff that I think is relevant for this topic. I read your guide (https://fragpipe.nesvilab.org/docs/tutorial_glyco.html) and I still have couple of questions. Below, when I refer to a section I refer to the numbers in the picture of the “PTMs” tab on the link above.
• How can I generate a .glyc file, could we get an example? Is it a tab-separated txt format with a header saying “glycanname hex galnax NeuAC” followed by lined describing these, like “glycan1 1 1 1”? • If I use such a .glyc file, what happens to/with the mass offsets entered at the MSFragger tab, are they considered? • What workflow would be compatible with the use of a .glyc file? • Would it be possible to make a general glyco workflow, where you can distinguish between O and N linked glycans by either enabling/disabling the “N-glycan Mode” in section 3 (of the link). If enabled, it is N-linked, if disabled it is O-linked. Also, one would need to change the AAs for localization in section 1 (again the link) and to switch to a relevant .glyc file. • Would it be possible to quantify these using IonQuant? Alternatively, could they be collapses into either all glycans of the same type throughout the dataset, or/and all glycans at the same position throughout the dataset? • Regarding section 4 (the link) how would this act on/with CID data? The “gap-masses” should be there, some of the breakdown products might be present as well, maybe even attached. • Last, and probably most importantly, how am I to interpret the output? So fare all I have is a global.glycoprofile.txt file. This is not really what I’m looking for, I would love for the identified glycans to be included in the general search so they could be treated as any other PTM and written in the output as such (preferably in the peptide.tsv and related output like MSstats ect.).
So, first of all thank you so much for your great work. I would love to be able to fully utilize it, but I really need some more information on how to do so and how to interpret the results. An example would be great. So, in summary, an example of a .glyc file would be great. Knowing if this can be used for both N- and O-linked glycans would be great. What workflows should be used for this, and how should I handle the MSFragger tab. Ideally, I would like for the glycans to be reported as all other PTMs included, this would also make the interpretation a lot easier. Lastly the oxonium ions, how to use these in CID, the description above describes these for HCD and that scores/penalty are involved, thus I'm wondering how this would act in a CID.
Kind Regards Martin
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MNTsnowman
dpolasky
Hi Fragpipe
I’m unsure of where to ask this question, so I’ll drop it here… If you prefer to take it by mail or something else, let me know and we can do that. I have been wanting to explore the glycoproteome of my samples, but I have no clue how to do it effectively and how to interpret the output. In Fragpipe V 17 I noted that the PTM tab is changed quite a lot with stuff that I think is relevant for this topic. I read your guide (https://fragpipe.nesvilab.org/docs/tutorial_glyco.html) and I still have couple of questions. Below, when I refer to a section I refer to the numbers in the picture of the “PTMs” tab on the link above.
• How can I generate a .glyc file, could we get an example? Is it a tab-separated txt format with a header saying “glycanname hex galnax NeuAC” followed by lined describing these, like “glycan1 1 1 1”? • If I use such a .glyc file, what happens to/with the mass offsets entered at the MSFragger tab, are they considered? • What workflow would be compatible with the use of a .glyc file? • Would it be possible to make a general glyco workflow, where you can distinguish between O and N linked glycans by either enabling/disabling the “N-glycan Mode” in section 3 (of the link). If enabled, it is N-linked, if disabled it is O-linked. Also, one would need to change the AAs for localization in section 1 (again the link) and to switch to a relevant .glyc file. • Would it be possible to quantify these using IonQuant? Alternatively, could they be collapses into either all glycans of the same type throughout the dataset, or/and all glycans at the same position throughout the dataset? • Regarding section 4 (the link) how would this act on/with CID data? The “gap-masses” should be there, some of the breakdown products might be present as well, maybe even attached. • Last, and probably most importantly, how am I to interpret the output? So fare all I have is a global.glycoprofile.txt file. This is not really what I’m looking for, I would love for the identified glycans to be included in the general search so they could be treated as any other PTM and written in the output as such (preferably in the peptide.tsv and related output like MSstats ect.).
So, first of all thank you so much for your great work. I would love to be able to fully utilize it, but I really need some more information on how to do so and how to interpret the results. An example would be great. So, in summary, an example of a .glyc file would be great. Knowing if this can be used for both N- and O-linked glycans would be great. What workflows should be used for this, and how should I handle the MSFragger tab. Ideally, I would like for the glycans to be reported as all other PTMs included, this would also make the interpretation a lot easier. Lastly the oxonium ions, how to use these in CID, the description above describes these for HCD and that scores/penalty are involved, thus I'm wondering how this would act in a CID.
Kind Regards Martin