Problems in analyzing my data, Chandni Kumar (Genecenter Munich)

[Kumar, Chandni]

Dear Sir or Madam,

hope you are doing fine in these difficult times.

I am a PhD Student of Dr. Franz Herzog (Biological Mass Spectrometry, Gene Center Munich) and I am analyzing my data with your Software.

Currently, I am running into the issue, that I cannot select my file to analyze the run. 

As far as I understood, after finishing my run I should have it in my "Analyses" column but there is no data to select.

Moreover, when I click on the Task it disappears.

Another thing is, that I do not know if I correctly installed it. Are there specific requirements to install it on Windows?

Unfortunately, I am not a bioinformatician and therefore I really have limited understanding of programing. 

I would be very greatful, if you could help me out.

Thanks in advance for your great help and wish you a nice day. Stay healthy!

Many greetings

Chandni Kumar

___________________________

Chandni Natalia Kumar

Doctoral Researcher

AG Herzog

+49 (0)89 - 2180 76695

ku...@genzentrum.lmu.de

Room: A 3.20

Genzentrum

Ludwig-Maximilians-Universität München

Feodor-Lynen Str. 25

81377 München

[gopalakri...@gmail.com]

I just had the same problem.

I can upload my mzml file with no problems. I created my glycopeptide hypothesis using a fasta file for the protein sequence and txt file for the glycan composition which worked. However, when I “search glycopeptide sequences” on the left it shows “finished” but I cannot see anything in the “analyses” column.

@Chandni, did you manage to fix this issue?

Also, I created my glycan txt file manually with just the following single line since this was a glycan I knew was definitely in my sample:

{Hex:7; HexNAc:2}       N-glycan

But is there a txt file I can download containing all the common glycans?

[Joshua Klein]

I'm getting the distinct impression that my UI design is obfuscating the task log, or that the task log is difficult to read.

gopalakrishnan1278, can you locate the log file for your glycopeptide search? It would be located in "<project directory>/task_dir/<task_name><timestamp>.log". If you can share it with me, it will help determine what might have happened. What kind of instrument and dissociation modes was your data acquired with?

Thank you,

Joshua Klein 

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Joshua Klein

[Joshua Klein]

Thank you. It looks like that single glycan was found on two peptides, but because the search space was so simple, the FDR estimation step couldn't separate targets from decoys. You might be able to work around this by making the search space more complex, or by raising the q-value threshold that is applied during quantification and protein assignment:

If you set that to 0.5 or 1.0, you should see your results. 

Another possibility is that your energy settings were too low for the glycopeptides in question and most of the fragmentation is dominated by peptide+Y ions, which are shared between targets and decoys. This is to be conservative, but I have a development version that I'm working on that can permute them to make them unshared. Looking at the energies you used, I don't expect that to be the issue, but if needed, I can accelerate the release of that feature too. 

Thank you,

Joshua Klein

On Mon, Aug 3, 2020 at 3:10 PM Pa Gopalakrishnan <gopalakri...@gmail.com> wrote:

Dear Joshua,

Thank you for your quick reply. The log file is attached. The instrument I am using is a Q Exactive with HCD (I used stepped NCE 27, 30, 33) and I converted the raw file to mzml using MSConvert's default settings.

Many thanks,

Gopi

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Joshua Klein

[Joshua Klein]

GlycReSoft uses a glycan model that can support arbitrary monosaccharide units expressed using IUPAC notation, provided that there is a common core motif. I really need to get around to documenting it better though.

If your sample is human or mammalian, you can use a pre-generated biosynthetic glycan database for N-linked and O-linked glycans:

The N-linked database is around 448 N-linked glycans and the O-linked database is 154 O-linked, though I'll grant you that the O-linked database is probably far larger than you're looking for. I use the attached glycan list when I'm looking for those N-glycans and a few smaller O-glycans. If you're looking for glycosaminoglycosylation, the linker tetrasaccharide and a few expansions of it are available in the "glycosaminoglycan linkers" option on the prebuild search space screen. Keratan sulfate is tricky because it both adds lactosamine units and sulfates, and none of the pre-generated lists include sulfation (save the GAG linker, as expected). 

I might be able to provide a better answer if you tell me about the sample you're analyzing.

Thank you,

Joshua Klein  

On Mon, Aug 3, 2020 at 8:56 PM Pa Gopalakrishnan <gopalakri...@gmail.com> wrote:

Dear Joshua,

Thank you so much for your quick reply – this is very helpful.

Your suggestion worked and I can now successfully see the glycans on the glycopeptides I was expecting J.

For the glycan compositions, I manually created my own txt file. Is there anywhere I can download a list of all glycans as a txt file to upload? I see that the glycans in the txt file need to be in the format:

{Fuc:1; Hex:3; HexNAc:2}              N-glycan

{Fuc:1; Hex:2; HexNAc:2}              O-glycan

etc

If I cannot download anywhere, do you have a txt file that you would be kind enough to share? I saw in your instructions the option of using the command line but I have no experience of doing this and think it would be too complicated for me.

Any advice on where to download or a txt file would be most appreciated.

Many thanks Joshua. Kind regards,

Gopi

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Joshua Klein

[Joshua Klein]

Viral glycans really depend upon the host expression system. I've done work with chicken egg grown viral data which could express NeuAc and NeuGc, so you'd need to use the mammalian lists for that. If they are grown in human cell line cells (e.g. HEK289), then the human glycan lists will be sufficient. That said, I've previously shown that viral N-glycans may be sulfated in varying abundance [1][2]. [2] is a preprint where I identify abundant sulfated N-glycans on glycosite 74 of COVID-19 from the data published with [3], and I don't include sulfation in those lists because it balloons the search space quite a bit, and it is hard to assign properly without really good precursor mass accuracy. I've attached the glycans used in [2] to this message.

Unfortunately, I left the ability to specify a custom protease out of the GUI. From the CLI, protease names and the regular expression that defines their cleavage patterns were interchangeable. I did add alpha-lytic protease to the enzyme name list recently, but obviously not recently enough for it to have been in the last release of the GUI. I'll make a new release tomorrow, if I can.

Thank you,

Joshua Klein

[1] Klein, J., & Zaia, J. (2020). Relative Retention Time Estimation Improves N-Glycopeptide Identifications by LC–MS/MS. Journal of Proteome Research, 19(5), 2113–2121. https://doi.org/10.1021/acs.jproteome.0c00051

[2] https://www.biorxiv.org/content/10.1101/2020.05.31.125302v1

[3] Watanabe, Y., Allen, J. D., Wrapp, D., McLellan, J. S., & Crispin, M. (2020). Site-specific glycan analysis of the SARS-CoV-2 spike. Science, 9983(May), eabb9983. https://doi.org/10.1126/science.abb9983

On Thu, Aug 6, 2020 at 6:36 PM Pa Gopalakrishnan <gopalakri...@gmail.com> wrote:

Dear Joshua,

 

Thank you for your email and apologies for my slow reply. You are very helpful.

 

Great you have the pregenerated glycan option and also thank you so much for sending the text file with all those glycans.

 

The samples I am looking at are viral glycoproteins (including bat coronavirus and recombinant). Are the pregenerated glycans ok for this?

 

Is there any way I can add a custom enzyme? One enzyme I am using is alpha lytic protease (cleaves after T, A, S and V).

 

Thank you again for all your help.

 

Gopi

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Joshua Klein