Hi Taleb,
Can we get a bit more of your targets list and the MS/MS spectra you expect them to match to? Your settings look okay, but with PRM the matching depends on the selected ion m/z of the MS/MS spectrum matching your targeted precursor m/z to within your "Method match tolerance" (i.e. 0.055 m/z).

So, you should be looking very closely at the precursor m/z for your target and the selected ion m/z for the MS/MS spectrum you hope to see Skyline extracting from. If you could provide screenshots showing those, that could be more informative.

You can also provide us the entire Skyline document (using File > Share to put everything into a .sky.zip file) and the raw data file (again in a ZIP file) on:

https://skyline.ms/files.url

Thanks for posting to the Skyline support board.

--Brendan

Hi Brandon,
Thank you for the quick response. I guess the problem is not from the m/z matching tolerance because the precursors are identified properly and I have manually verified that the fragments are present within the mass tolerance range. I guess the problem is that the MSMS scans for some reason not imported/read because when I click on the precursor chromatogram, the pop-up full-scan spectrum only contains MS scan. If I instead select "DIA" as the acquisition method, instead of "targeted", and import the same raw file, the full-scan correctly includes one MS scan and one MSMS scan.

I have uploaded the following files for your review:

The zipped Skyline document: PRM_BTubulin.sky.zip

The Masslynx raw file: 20200517_PRM4_BTubulin_5pep.raw.zip

Thank you so much for your time!

Taleb

Taleb,

You should go to:
Settings > Transition Settings > Instrument
and change "Method match tolerance m/z" to a higher number, such as 0.2.

Currently that is set to 0.055, which is not high enough for the m/z's in your raw file to agree with what they are in Skyline.

For instance, you have a peptide in Skyline whose precursor m/z is 830.4512, but the MS/MS scans in the raw file say that their precursor m/z is 830.345.

(Also, choosing "DIA" instead of "Targeted" would work too.)
-- Nick

Hi Nick,
Thank you so much!!! The issue was resolved.

Taleb

Nick's analysis was essentially what I was trying to suggest that you do yourself, Taleb. When MS/MS spectra are not matching to your targets, you should begin by looking at the target m/z values in Skyline and comparing them with the MS/MS spectra precursor or isolation m/z values (which you specified in your instrument method). There are a number of tools you can do this with, but if you lack a good tool you can use to look at the MS/MS precursor or selected ion m/z values, you can always install ProteoWizard (http://proteowizard.sourceforge.net/download.html) and use the tool SeeMS which comes with that installation.

If you can't report both the target m/z and the method m/z (what the quadrupole was set at) that are failing to match, then you haven't looked deeply enough at what you are trying to achieve.

Given what Nick reported, it is still worth asking yourself why you ended up with a method that set the quadrupole to 830.345 for a target with a precursor m/z of 830.4512. Is this just a typo? Was the method supposed to specify 830.45 and somehow an extra 3 got added after the decimal point? Or were you aiming for something else?

Just setting your "Method match tolerance m/z" to 0.2 or using DIA as your method is not necessarily the right answer, though it may be what you need to salvage this dataset. The easiest way to avoid issues like this would be to use Skyline to export your isolation lists for PRM.

Glad you were able to resolve the issue and use your data. Hope this discussion was helpful.

--Brendan

Hi Brendan,
I should have been more clear and thorough, in explaining all the details:
- With your suggestion, I checked the targeted vs. acquired m/z values for the precursor/product. The m/z values were accurately exported from Skyline method into the Masslynx acquisition method (via isolation list). Also, the m/z values were acquired with a good mass accuracy in the MS & MSMS scans (e.g. a few ppm of error as checked within Masslynx). But I did not realized that the actual quadrupole set values for precursor ions were off by 0.11 Da for all precursors until Nick pointed out. This seems to be Masslynx issue and I will contact Waters support for it.

I also need to clarify that the product ion chromatograms still do not show up for the RAW file that I sent to you, even when a 0.2 Da is selected as the method tolerance. But with another file all precursor/product peaks are present (both injections were acquired with the same acquisition method and quadrupole set values are off for both). I am not sure what is the issue with that first file.

I have shared the second RAW file for your review: 20200517_PRM5_BTubulin_5pep.raw.zip

Thank you for your help,

Taleb

I am not sure I understand your new question. Can you send us a screenshot of what you are looking at?

By the way, when I try to extract chromatograms from the new .raw file that you uploaded ("20200517_PRM5_BTubulin_200ms.raw"), Skyline abruptly exits (an access violation inside of MassLynxRaw.dll). That does not sound like the symptom you are describing, so could you try uploading that file again (to a different name) and I'll see if there's a difference?

-- Nick

I would also like to note that just making the change Nick suggests would not be expected to automatically give you fragment chromatograms in PRM. You must reimport the data for Skyline to extract those. The target to MS/MS matching happens during import time and chromatograms are only created from MS/MS which match the targets in the document.

Have you tried either: 1) removing the original file, saving the document, and importing it from scratch? 2) using Edit > Manage Results, select the problem file, click Reimport and click OK?

Thanks for all your help in understanding the issues you are encountering.

--Brendan

Hi Brendan and Nick,
You are right! Although I removed and re-imported files upon changing setting each time, I did not save Skyline after removing the chromatograms. Please ignore my earlier message. It is all fine now except that I need to find while Masslynx set the quadrupole as a m/z different than the one set in the acquisition method.

Thank you very much for your patience!!!

Best wishes,

Taleb