method match tolerance kbagramyan  2019-12-17
 

Hi Brendan,

I am glad that I found the question that I wanted to ask you too. We recently performed an MRM analysis using 6495 LC-MS Triple Quadrupole (Agilent). We optimized the MRM transitions for the quadruply charged precursor ions of the light and the heavy peptides. Here, are our transition settings used in the method: 545.3 (precursor, +4) and the transitions are 509.0 (b, 4+); 640.0 (b, 3+); 678.3 (b, 3+). The Skyline recognized the two of transitions such as 508.5155 and 639.67. The third transition of 678.3 in our method was 677.6849 in Skyline transitions giving 0.6151 Da difference. Unfortunately, this was the most intense peak but wasn't accepted by Skyline because I couldn't extend the method match tolerance above 0.6.
The data look very good and I was wondering if there is a way to extend the method match tolerance, so I can include that transition for accurate quantification?
I hope I was clear and was able to describe the problem.
Thank you in advance,

Karine

 
 
Brendan MacLean responded:  2019-12-17

Hi Karine,
Sorry, I don't think we will expand the method match tolerance to cover this mistake. You will be much better off in the future, if you use Skyline to generate the methods for which you expect to import data into Skyline. Skyline has been available for over decade now and most of this flexibility to deal with transition calculation and manual entry errors described in the Existing and Quantitative Experiments tutorial was built to support methods created before Skyline existed. That method match tolerance got as wide as 0.6 was an artifact of another method development researcher (at the ISB) requesting this for her research. I think it is already way too wide when a quadrupole may only be filtering 0.7 m/z total.

I can see how measuring lighter than the monoisotopic mass on a multiply charged fragment may still give you good signal. The researcher who requested the 0.6 tolerance was researching whether it might produce more signal to center a triple quadrupole over a lighter mass and so filter more of the isotope envelope.

At this point, however, I would recommend you try to work out some kind of modification on your peptide which shifts the masses into the range accepted by the method match tolerance. Skyline has very flexible support for peptide modifications. Or if you fail that, you could simply use a small molecule transition list to specify exactly the masses you measured.

Hope this helps. Good luck with getting your runs as acquired into Skyline in a way that you are happy with the end result analysis.

--Brendan

 
kbagramyan responded:  2019-12-17

Hi Brandon,
Thank you for the detailed and informative response.
Best,
Karine

 
kbagramyan responded:  2020-03-11
Title: adding modifications to peptide sequence

Hi Brandon,
Could you please tell me how I can add modification to one amino acid only in the peptide sequence that doesn't affect the second amino acid. Here is the sequence: TRIDEANQRATK.
It contains two arginines in the N and the C-terminal parts of the peptide. A heavy peptide that we are using in our measurements should have N-terminal arginine modification with [13C]6H12[15N]4O. The second, C-terminal arginine remains unmodified. When I am trying to add modification into the Skyline through the peptide settings, it keeps modifying both arginines even after defining that the modification has to be at the N-terminus only.

Thank you,
Karine

 
Nick Shulman responded:  2020-03-11
Karine,

You can right-click on a peptide in the Targets tree and use the menu item "Modify" to apply modifications or to remove modifications from an individual amino acid.

You might want to make this particular modification into a "explicit" modification, which means that it appears in the list at "Settings > Peptide Settings > Modifications", but it does not have a checkmark next to it. Explicit modifications are ones that Skyline does not apply to a peptide unless you explicitly ask for it, either by using the Edit Modifications dialog, or by specifying the modification mass in square brackets in some place such as "Edit > Insert > Peptides".

If you would like to learn more about static, variable, and explicit modifications, you can take a look at Webinar 10 "Working with modifications in Skyline":
http://skyline.ms/webinar10.url
-- Nick
 
Brendan MacLean responded:  2020-03-11
Also covered in the second half of the Existing and Quantitative Experiments tutorial under Adjusting Modifications Manually:

https://skyline.ms/_webdav/home/software/Skyline/@files/tutorials/ExistingQuant-1_4.pdf#page=19
 
kbagramyan responded:  2020-03-12
Thank you!
That was very helpful!
- Karine