Cant see the chromatograms

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Cant see the chromatograms mostafa khan  2017-10-16 11:49
 
Hi,

For some reason I am not able to see the chromatogram for any of the samples for one of the compounds, while I see them clearly with the rest of the compounds. I changed the product ions in the transition list, yet I cant see them, although I see them clearly in xcalibur. What am i doing wrong?

Thank you in advance.
 
 
Brian Pratt responded:  2017-10-16 11:59
Hi,

Do the precursor mz values in the Skyline document match the ones you're looking at in xcalibur? I'm guessing there's some confusion around the isotopic labeling, perhaps?

Brian
 
mostafa khan responded:  2017-10-16 12:22
Hey Brian,

Yes, so selected the value from xclaibur then I put them on skyline.

Mostafa
 
Brian Pratt responded:  2017-10-16 12:56
Hi Mostafa,

Can you provide:

The transition list file you used to import these values into Skyline
The resulting Skyline document, using the File | Share menu item in Skyline
The raw data file

If you can upload all that to https://skyline.ms/filecontent/home/support/file%20sharing/begin.view? we can sort this out, I'm sure.

Brian
 
mostafa khan responded:  2017-10-16 14:07
Hey,

I have uploaded the RAW file and also the skyline document.

Mostafa
 
Brian Pratt responded:  2017-10-16 14:33
Thanks - it would also be useful to see whatever you used to create the Skyline document. You probably have a spreadsheet or something that you used with the Import Transition List feature? I want to make sure that import step worked properly.

Thanks

Brian
 
mostafa khan responded:  2017-10-17 07:53
Hey,

Here is my transition list-

Molecule List Name    Precursor Name    Precursor Formula    Precursor Charg Product Charge Precursor m/z    Product m/z
15:0-18:1(d7) PC    15:0-18:1(d7) PC    C41H73 H'7 NO8P    1        1 753.6125 184.0725
15:0-18:1(d7) PE    15:0-18:1(d7) PE    C38H67H'7NO8P        1        1 711.5657 570.5507
18:1(d7) MG    18:1(d7) MG    C21H33 H'7O4    1 1 386.3263 303.7459
15:0-18:1(d7) DG    15:0-18:1(d7) DG    C36H61H'7O5        1        1 610.5403 368.3149

Mostafa Khan
 
Brian Pratt responded:  2017-10-17 08:01
Can I get that as an actual file, please? I prefer to be working with exactly what you were working with, and this has undergone some changes in the copy+paste+email chain.

Thanks
Brian
 
Brian Pratt responded:  2017-10-17 08:11
Also please provide the entire file, not just selected parts (all I see here are the heavy labeled versions). It's more informative if I can start where you started.

Thanks,
Brian
 
mostafa khan responded:  2017-10-17 08:14
Here is the excel file

Mostafa
 
Brian Pratt responded:  2017-10-17 09:13
Hi Mostafa,

There's something amiss here, certainly - Skyline issues these warnings:

"Error on line 3: Precursor m/z 386.3263 does not agree with value 364.3439 as calculated from ion formula and charge state (delta = -21.98243, Transition Settings | Instrument | Method match tolerance m/z = 0.055). Correct the m/z value in the table, or leave it blank and Skyline will calculate it for you."
and
"Error on line 4: Precursor m/z 610.5403 does not agree with value 588.5579 as calculated from ion formula and charge state (delta = -21.98241, Transition Settings | Instrument | Method match tolerance m/z = 0.055). Correct the m/z value in the table, or leave it blank and Skyline will calculate it for you."

Working back upstream to understand this mismatch in formula and reported m/z in whatever tool produced the transition list will probably clear things up.

Cheers

Brian
 
mostafa khan responded:  2017-10-17 10:09
Hey Brian,

So yes I see those, its because of the precursor formula which needs to be changed as these are Na adducts. I usually fix these before submitting the transition list. Once all is ok, then I upload the raw files, and the rest u can see that I was unable to see chromatograms.

Mostafa
 
Brian Pratt responded:  2017-10-17 10:22
Hi Mostafa,

All you need to do, then, is declare the adduct in the transition list. This works fine, for example:

Molecule List Name,Precursor Name,Precursor Formula,Precursor Adduct,Precursor Charge,Product Charge,Precursor m/z,Product m/z
15:0-18:1(d7) PC,15:0-18:1(d7) PC,C41H73 H'7 NO8 P,[M+H],1,1,753.6125,184.0725
15:0-18:1(d7) PE,15:0-18:1(d7) PE,C38H67H'7NO8P,[M+H],1,1,711.5657,570.5507
18:1(d7) MG,18:1(d7) MG,C21H33 H'7O4,[M+Na],1,1,386.3263,303.7459
15:0-18:1(d7) DG,15:0-18:1(d7) DG,C36H61H'7O5,[M+Na],1,1,610.5403,368.3149

Let me know if that helps.

Brian