Thank you for sending me your .sky files and then sending me that .sky.zip after your files were fixed.
When inserting a transition list, if Skyline cannot figure out how two precursors which are supposed to have come from the same molecule got the m/z's that they did, Skyline gives one of them a mass offset and assigns it the label type "heavy". The bug is that Skyline is not checking whether "heavy" is actually a valid label type in your document.
| Molecule Name |
Precursor m/z |
Product m/z |
Precursor Charge |
Product Charge |
| Uric acid |
169.04 |
152 |
1 |
1 |
| Uric acid |
167.02 |
124.1 |
-1 |
-1 |
When you do that, Skyline thinks that the positively charged precursor is a heavy isotope with the adduct [M2.0211+] heavy.
The easiest way to prevent Skyline from thinking that is a heavy precursor would be to add a "Label Type" column which you fill in with "light".
| Molecule Name |
Precursor m/z |
Product m/z |
Precursor Charge |
Product Charge |
Label Type |
| Uric acid |
169.04 |
152 |
1 |
1 |
light |
| Uric acid |
167.02 |
124.1 |
-1 |
-1 |
light |
Skyline still thinks the positively charged precursor has an adduct whose mass is 2.0211, but it no longer is calling that "heavy".
If you want to get rid of that mass offset adduct you could tell Skyline about the M+H and M-H precursor adducts:
| Molecule Name |
Precursor m/z |
Product m/z |
Precursor Adduct |
Product Charge |
Label Type |
| Uric acid |
169.04 |
152 |
M+H |
1 |
light |
| Uric acid |
167.02 |
124.1 |
M-H |
-1 |
light |
Skyline now thinks the positive precursor has a mass offset of 0.00545, which is a lot closer to zero.
If you want to get rid of that mass adduct completely you could add 0.00545 to the negative precursor m/z, or subtract 0.00545 from the positive precursor m/z, or something in the middle.
| Molecule Name |
Precursor m/z |
Product m/z |
Precursor Adduct |
Product Charge |
Label Type |
| Uric acid |
169.04 |
152 |
M+H |
1 |
light |
| Uric acid |
167.02545 |
124.1 |
M-H |
-1 |
light |
We are looking into whether there is a way to make Skyline less strict about adding these mass offsets for really small mass differences.
If you specify chemical formulas and adducts for these molecules instead of m/z's and charges, Skyline will calculate the appropriate m/z's and it would probably be easier.
You can learn more about adducts in Skyline here:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=adduct_descriptions
-- Nick
2.0211_heavy.png
2.0211_light.png
00545.png
NoMassAdduct.png