MS1 Quant with t-SIM partially solved, still questions on skyline

MS1 Quant with t-SIM partially solved, still questions on skyline Tobi  2018-06-22

Dear the whole Skyline Crew,

hope you had a good time in San Diego, I sadly could not join, so it would be nice if I can please ask you something here.

We want to do absolute peptide quantification, at first without any MS2 data or library (I read the older forum posts on this topic, hope I didnt miss anything). We measured 5 BSA Peptides of a simple solution without any background or standard with both a t-SIM only and a Full Scan t-SIM aquisition on thermo q exactive hf. Adding the peptide target list and loading the thermo raw files into skyline works fine and we get the t-SIM MS1 data.

The Problem is to get the Full Scan MS1 data with skyline from thermo raw files, which contain both t-SIM and Full Scan MS1 data (only t-SIM data gets displayed). A solution we have for now is to use MSConvert and filter the msLevel 1 into an mzXML file. When loading the mzXML file we do see the Full scan data, which is nice, but it takes a lot of time and resources.

Is there a way to selectivley display both the t-SIM and the Full Scan MS1 data with skyline from the original raw file directly? Or do you know any other workaround, like making a exact copy of the raw file just without the t-SIM data?

The second small issue, could you please help me with an opinion on if to use the high-selectivity extraction feature (halving the extraction width), especially for high resolution t-SIM data? At least for FullScans on samples with complex background, the area to background ratio gets improved, but the detected m/z changes slightly. For now, I am just not sure if the accuracy of the picked peak remains as high as without high-selectivity extraction.

For the end, I would just mention the observation, that displayed peak area replicate comparisons are sometimes shown as a line graph even if bar graph type is selected, just for your information.

Skyline seems pretty nice and I wish you continued succes with it in the future.

Best regards,

Brendan MacLean responded:  2018-06-22

Hi Tobi,
I started down the path of making this possible years ago, but then no one really seemed to be waiting to get it. So, I never quite finished the work it would take to make this possible within a single replicate in a single document.

I am afraid the solution is more involved than we can offer to do for you in the very near future, but your request at least shows some new interest in having this capability, and that certainly raises our perceived value of doing the work.

For now, though, I am afraid we can't offer any solution simpler than what it sounds like you have worked out.

Sorry for that, but thanks very much for letting us know of your data set that would benefit from the flexibility to extract MS1 chromatograms from both MS1 survey spectra and more targeted SIM spectra.


Tobi responded:  2018-06-25

Dear Brendan,

thank you very much for the very quick response, I can fully understand your explanation and I know skyline is used for many different projects, so no need to be sorry.

Just want to add, for some users it might be sufficient to just being able to choose which type of MS1 data to load into skyline, instead of loading both types of MS1 data (Full Scan/t-SIM) in one document. It would enable just making a copy of the raw file and loading both identical raw files into two different instances of skyline, therefore enabling to read out both data types straightforward without any additional software.

So far we are happy that it works, and might even find a way to circumvent the usage of the large mzXML files.

Best regards,

szabo zoltan responded:  2018-07-30

Hi Brendan and Tobias,

I wanted to request a similar thing. Extracting precursor from MS2 scans (also from SIM scans in the presence of full scan MS1).
I thought it may be easily incorporated by defining 2 kind of precursors (eg. 'p' and 'p2' or 'q' in ion types), but according to Brendan's response, may be not.
My workaround presently is defining it as small molecule transition, adding the precursor mass as transition too. So if manual 'freehand' add transition feature would be available for peptides too, it would also simplify the process a little bit (and may be useful to integrate special losses, marker ions etc.).

PS. I did not want to start new request, as it seems to be the same, and hopefully it may help for Tobias, too.

Sorry, I tried with SIM, and did not work. So I may request the other problem, precursor from MS2 (eg. PRM) scan.

Best regards,

AnnieWang responded:  2021-01-26

Hi Tobias,

How do you get the full scan/scan type panel displayed?
Can the current version of skyline distinguish full scan MS1 and tSIM MS1?

Thank you,

Tobi responded:  2021-01-26

Hi Annie,

I checked with the current skyline-daily. The Scan type drop down window should now show just one type of MS1 after clicking on some point of a chromatogram to open the view on the corresponding scan.

The chromatogram extraction is same as before. If your raw data contains just one type of MS1 it should be extracted all the time. If a thermo raw file contains both full scan and tSIM Skyline will read only the SIM. If a file containing both is converted (thermo raw to mzML by MSConvert) and loaded it should extract only the full scan.

For peptides we do not use SIM anymore but prefer DIA and PRM. For unusual cases that work only on the MS1 BoxCar might be worth a look but I have no personal experience there.

Hope it helps,
best wishes,