Exporting scheduled MRM method to *.exp Masslynx

Exporting scheduled MRM method to *.exp Masslynx per larsson  2024-02-01 09:05

Dear Skyline support,
I run lipidomics using waters xevo tqxs. Managing the many MRMs in the Masslynx software is to time consuming (adjusting RTs manually) so I am trying to learn skyline. My main objective is to be able to write all MRM experiments into a *.csv file that can be used as a compound database. I can then use skyline to build the *.exp method for each application and update the RTs in excel.

I manage to insert transition list with the parameters needed in a way that seems correct into Skyline but I have problems to get all needed settings exported to the *.exp file.


  1. The problem is that when I export method I am not able to get the right polarity set for each MRM in skyline into the *.exp file, they will al be ES+ even if listed as ES- in my Skyline file.
  2. I have the retention time and retention time windows in Skyline but am unable to get export this to the *.exp file the start and stop time will be at the same value.

If I can find a solution to these problems I would be able to write and modify all my methods from templates in excel and export them very quickly as needed to Masslynx. Changing RT times and windows would be a very simple task in excel and making selections for MRMs for a new application would be very simple as well. To make this work I need to find the solution to export the scheduled RTs and the polarity for all MRMs from skyline to masslynx.

I am using Masslynx 4.2 SCN 1045 and Skyline and run this on the workstation used for controlling the instruments.

In case any one can help me I attach my csv file that I use to import into skyline, pasting into "insert transition list"
I attach my skyline file
I attach my .exp template for the export
I attach a raw file with results from the lipids in the template

Would be extremely nice to find a solution and I am think there should be lots of other users that would have use of this solution.
Excel->Skyline-> Masslynx for method setup would be great


Brendan MacLean responded:  2024-02-01 11:19

Hi Per,
Looking at our code, it looks as though we do not presently pass an ion mode to the DLL supplied to use by Waters for method export:


The TransitionList argument is a text string in the same format you would get out of File > Export > Transition List which does not include an ion mode specifier.

I am curious whether you might somehow supply Skyline with a template method that implements the ion mode? Are you looking to create a mixed mode method with both positive and negative? Perhaps if you want only negative, you can specify that somehow in the template and the VerifyESkyline.dll would preserve it?

I have begun a discussion with Waters about whether we might be able to support this with the current VerifyESkyline.dll or wether we would need a new DLL to be able to specify ion mode individually for each transition.

Thanks for bringing this up. Sorry it doesn't seem to be working already.


per larsson responded:  2024-02-01 13:26

Hi, Big thanks for investigating. The masslynx template i use is the .exp i used originaly to run the lipids. I tried to recreate the method from *.csv to skyline to masslynx. So the exp template have es+ and es-. I could not find any additional column in skyline import more than charge for setting ion mode. I want to run both es+ and es-. This is part of why it is so vital to get the rt windows right so that I can avoid polarity switch in the mrm cycle. Some of my lipids are close so i need to really time this right. I also tested the .exp file supplied in one of your tutorials and mrms created from lipidcreator plugin. Exported methods are always es+ in masslynx. Let me know any suggestions you i can test to assist in troublshooting.

Brendan MacLean responded:  2024-02-01 14:12

Can you give us an example of a text file transition list that contains the information you want and which is easy for you to load into MassLynx? This would ideally influence how we implement File > Export > Transition List, and what we send to VerifyESkyline.dll to create the .exp file.

I have attached what Nick sent me from a File > Export > Transition List for your targets. This is essentially what we are currently sending to VerifyESkyline.dll (from Waters) to create the .exp file. Yes, it has no polarity information. So, I think a good place to start is to update this format to contain a polarity column. I am just not sure where it should go in this format or what its column header name should be.

Thanks for your feedback.

per larsson responded:  2024-02-02 05:05

An example import .csv file is uploaded I used the molecule setting for the import as my MRMs are lipids so columns heading are a little different, you can see them in the .csv file I uploaded.
I have setup and experiment where I have optimized the retention time window for each MRM for as efficient acquisition as possible to maximize dwell times. Some MRM windows are a lite bit wider and some are a bit shorter. If there is one MRM in ES+ and the next is ES- i will try to time it so the MRM don't overlap. It is important for me to be able to choose my selected retention time windows in an easy way. This was my issue with waters own Quanpedia software that any automatic update feature of retention time resets my adjusted retention time windows to the same value for all MRMs.

For learning the software, I made a simple list of just a few standards for testing. I used an *.exp file created for the data acquisition in masslynx and write the same parameters into the .csv file in the Skyline format (molecule setting was used not peptide). I can then import .csv file into skyline and it seems to get everything right (MS1/MS2/CV/CE/Charge/RT/RT_window) are all registered. Acquired RAW file seems to work well I get all my MRMs into Skyline as expected with correct RT and RT windows and charge (this I also uploaded). As far as I see import to skyline work fine it shows correct RT and RT_window for imported data and in the preview for the export .

The problem is that values are not exported to the *.exp file. Only MS1/MS2/CE/CV is exhorted not the polarity(charge) or the retention time window. To manually add these for a large nr of MRM is not what I want to do.

From the values in Skyline of charge RT and RT_window the following settings need to be transferred to the waters instrument *.exp file
FunctionIonMode,ES Mode
(in skyline i charge 1 means it should be positive and -1 it should be negative)
(from skyline RT and RT_window the start time and end time can be extracted)

I am using a very simple method to test the import and export but for my real methods I need to get this working without manually adjusting polarity and RT windows for all exported methods. Given the tutorials have scheduled methods exported I assume it has worked at some point. Either I am doing it wrong or the function needs to be fixed. Lets see if we can figure it out. Has anyone with a waters xevo tqxs recently been able to export a scheduled MRM method?

It is a bit annoying that the waters .exp file is composed as a text file an not as variable table that would be very easy to import and export to. Given I have not made any error I hope that Skyline and Waters can manage a way to export the required data from skyline to a .exp file that is working with masslynx and Xevo TQXS. I will be happy to help in any way I can but unfortunately I don't know much about programming and have just started to work with skyline. Since there is just a limited nr of parameters needed to be modified to export new MRM exp I am hoping that it can be solved.

*Compound Name

UPDATE: Using the everify.exp template from skyline tutorial I can export shedulted MRM but only if I use 1 min retention time window.


per larsson responded:  2024-02-20 05:59
I have watched the small molecule webinars and also used the same data that was uploaded for testing. I am confused since the tutorial is exporting scheduled methods using the Xevo instrument so apparently this was tested by Will Thompson. However in the tutoral it is never shown that there is any information for the scheduled MRM in the exported files.

It is most frustrating that I can import all the information into skyline using the insert transition list but then not get it exported to the *.exp file that I can use with the instrument. In molecule setting, if I use predicted retention time and time window of 1 min I can get this to be exported but only if I used 1 min or 2 min time window, if it is less than 1 min window then start and stop time will be the same value in the *.exp file and I will have to manually set start and stop for each MRM in masslynx.

This is the Skyline parameter that needs to be transferred to Masslynx *.exp parameter that I can not get to work.

"Precursor Charge" = "FunctionPolarity" (it would be good to be able to export method using ES+ and ES-)
"Explicit Retention Time" and "Explicit Retention Time Window" = "FunctionStartTime" "FunctionEndTime"

I hope I have made the issue clear and that Skyline developers and Waters could collaborate to find a solution. If by any chance anyone have ben able to export Explicit Retention Time Windows set in skyline to Masslynx *.exp file please let me know. I would expect there to be lots of users with the xevo using skyline so I would assume lots of users would appreciate this to work.
I attached a print screen showing the imported parameters and that they seem just right in skyline just need then exported to the format used by masslynx in the .exp file.

Sorry for the long texts, but would really love to se a solution to this and make skyline the main software for our method development and sample processing given the nice features it has, exporting of methods and importing of data must work to make this possible.
Rita Chupalov responded:  2024-02-21 13:19
Hello Per,
we recently lost the guy who wrote most of the vendor-specific method import/export code, so I am taking over this area. Unfortunately I did not work with Waters methods yet, so it might take some time to install their software and figure out how to pass this parameter to the .exp file. Would you be open to have a brief Zoom meeting next week so that we can walk through your workflow and I get better understanding of the problem? Meanwhile I'll install the MassLynx and familiarize myself with it and the software interface between MassLynx and Skyline.

per larsson responded:  2024-02-22 13:34
Hello Rita,
This would be great. I really appreciate that you will try to see if you can help me. I have access to workstation used for data acquisition and latest scn1045 for masslynx targetlynx in case this would be needed. Let me know when you have a time.
Rita Chupalov responded:  2024-02-23 13:02
to set up this meeting could you e-mail me at ritach (at) uw.edu? I don't want to post the zoom link to this page, since it's public.
Thank you.