Hi Amir,

you really need to provide more information in order to get assistance. What kind of data analysis did you do on the MS? ie. what type of scan? What are the settings you used for MSConvert? What was the output file format? What are the settings you used for MaxQuant?

I'ts unclear if MaxQuant is not reading your data, or not giving the "desired outcome."

I see no reason you cannot use data from the Bruker as input to MaxQuant. I'll assume you did a DDA analysis and if you convert that to MGF using MSConvert, then MaxQuant will be happy.

Good luck,

Peter

Hi Peter,

Thank you so much for your kind support. I apologize for the lack of information and will do my best to answer your questions. I want to identify my proteins and perform LFQ analysis. I have tried different settings on MSConvert to convert the raw data generated by Bruker (which is in a .d folder) to .mzML and .mzXML formats. Additionally, I have exported the compounds from DataAnalysis Software as .MGF files, but MaxQuant does not recognize them. The settings I used in MaxQuant were the same as those recommended on the official MaxQuant website for LFQ analysis. Please find attached the standard parameters I used for mass spectrometry methods and peptide identification.

Best regards,

Amir

Nikhil Dev Narendradev

Hi Nikhil,

Thank you so much. I will try and inform you if it works.

Nikhil Dev Narendradev

I tried Fragpipe and this software also could not recognize the data.

Hi Amir,

when you say it "does not recognise" the data, what do you mean? Do you get an error message? What is the message? It's odd that two tools fail. I think your problem lays with the data, or the conversion.

What does the TIC look like? Do you have peaks? Can you see MS/MS fragments in the data? How long is the LC run? How big are the files at the end of the run? How big are the mzXML or mzML files?

Can you attach one of the MGF files so we can have a look?

Hi Peter,

I selected the Load folder and browsed to the .d folder. I then checked the Recursive tick and clicked Ok. However, the software did not recognize any files, meaning no files appeared in the list (please see Fig. 1). When I converted the .d folder to .mzML or .mzXML, MaxQuant was able to recognize the file, but starting the evaluation resulted in an error (please see Fig. 2). I have the peaks, MS/MS fragments, and the LC runs for 55 minutes. You can see the peaks in Fig. 3 and Fig. 3-1. At the end of the run, the files are not very large in size. The .mzML files are also not big. Please see the content of the raw data generated by the system in Fig. 4. Kindly find .MGF file attached. I cannot say thank you enough for your help. :)

Hi Amir,

okay you've got some peaks, but not heaps. That is likely the underlying issue here, you do not have a lot of data for searching. I had a look at your MGF and there are clearly masses in there that "look" like real peptides. I did a search using Mascot, using your MGF file, and some generic settings as I do not know anything about the sample. I was able to get matches with ~10 proteins, above the significance score. I looked at some of the peptides and those were a good match in my view. But there are hardly any peptides.

My feeling is your issues are related to the actual data that you've acquired, not the data conversion, and not the use of MaxQuant or FragPipe.

There are peptides in your sample, they do match with proteins in SWISSPROT, but the number of significant hits is very low. This, along with your clearly very low intensity TIC, all leads me to believe that your issues are related to how much sample you are putting on the MS itself. ie. load more if you can. if you can't you need to find a more sensitive MS. I realise that may not be possible for you.

I hope that gives you something to work with.

Peter

Hi Peter,

I warmly appreciate your kind support. I will try to load more sample on the MS.

Have a good one. :)

Amir

Hi Amir,

I think I know the issue with your data because am currently at the same stage of analysis. I used to get the same error when I run my samples which were converted to MZXML formate using MSConvert tool in maxquant. the thing I discovered that Maxquant doesn't recognize any MZXML files except the ones created by OpenMS (TOPPAS) tool which have a specific option to make it recognizable by maxquant ''Force_MaxQuant_compatibility''.

To make it easier please follow step by step;

1) open Galaxy Europe (a free online tool that provides graphical access to multiple genomic and proteomic tools as OpenMS)using the link ------https://usegalaxy.eu/

2) create an account

3) search for FileConverter tool from the toolbars on the left of the page, and click on it

4) Then load the files that you wish to convert under input files to convert, (note you can load all the files at once)

5) then, specify the output type formate to mzxml, and under advance options set yes option to [mzXML output only] Make sure that MaxQuant can read the mzXML and set the msManufacturer to 'Thermo Scientific'

6) and then click run tool , it will take few mins and your desired converted files will appear on the right bar on the page, then you can download the converted files one by one.

one more hint; when setting the parameters on maxqaunt keep the instrument as orbitrap and make sure to run the analysis using a PC with a high processor and space or cloud space as the quanting can take upto days of continuous run

and that's all. all the best :)

Dina Osman Hi Dina,

Thank you so much for your kind response. I will follow the procedure you explained and will update you on the final result.

Have a good one. :)

Best regards,

Amir