Skyline Small Molecule Targets
The Skyline Targeted Proteomics Environment provides informative visual displays of the raw mass
spectrometer data you import into your Skyline documents. Originally developed for proteomics use,
Skyline has been extended to work with generalized small molecules. There are many tutorials available
that will help you use Skyline for various types of analysis (SRM, MS1 Filtering, DIA, Targeted MS/MS
etc). This tutorial concentrates on the differences in using Skyline for targeting small molecules.
In this tutorial, you will build an MRM assay for a group of Methionine-pathway compounds.
Skyline aims to provide a vendor-neutral platform for targeted quantitative mass spec research. It can
import raw data from the instrument vendors Agilent, SCIEX, Bruker, Shimadzu, Thermo-Scientific and
Waters. The ability to import data across various instrument platforms greatly facilitates cross-
instrument comparisons and large multi-site studies. This remains equally true in using it to target small
molecules, as it has been for years in the field of proteomics.
Getting Started
To start this tutorial, download the following ZIP file:
[Link]
Extract the files in it to a folder on your computer, like:
C:\Users\bspratt\Documents
This will create a new folder:
C:\Users\bspratt\Documents\SmallMolecule
It will contain all the files necessary for this tutorial. Now start Skyline, and create a new empty
document.
Importing a Small Molecule Transition List into a Skyline Document
The easiest way to get a small molecule transition list into a Skyline document is to start with an empty
document and use the Edit > Insert > Transition List menu item. [Note: the File > Import >Transition List
menu item also works, so long as your data has column headers which are recognized by Skyline. For a
list of recognized column names, press the “Help” button in the Edit > Insert > Transition List window.]
At a minimum, Skyline needs to know the charge state and either the ion formula or m/z for each
precursor and product. If no product ion information is present in a transition list, it is assumed to be a
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list of precursor targets. Repeated precursor information with different product information is assumed
to indicate multiple transitions of a single precursor, just as with peptides.
A note on ion formulas and adduct descriptions
In proteomics applications Skyline can safely assume ionization by protonation. So, all that is needed to
describe a charged peptide is its sequence and charge state. For generalized small molecules, however,
ionization can be achieved by almost any means (sodium gain, hydrogen loss, etc.). Such ionization
modes can be specified in Skyline using adduct descriptions of the style used at
[Link] e.g “[M+Na]”, “[M-2H]”,
“[2M+ACN+H]”, etc.
Adduct descriptions can also be used to indicate that a molecule is isotopically labeled. For example, the
adduct “[M3Cl374H2-Na]” indicates that the molecule has three Cl atoms replaced by 37Cl, and four H
atoms replaced by deuterium, and the molecule is ionized by loss of Na.
For molecules where the formula is unknown, a mass shift can be indicated by adduct. For example,
“[M(-1.23)+H]” indicates that the molecule’s mass is 1.23 AMU less than its unaltered form, and the
molecule is ionized by hydrogen gain.
For molecules where the means of ionization is unknown, an adduct can indicate just a charge. For
example, “[M+3]” indicates charge state three. The m/z value will be 1/3 of the molecule mass. While it
is possible to describe your transition list completely in terms of m/z values for both precursors and
products, without a chemical formula Skyline cannot provide isotopic distributions so chemical formula
and adduct descriptions are preferred.
Transition list insert
To begin creating your first Skyline document that targets small molecules, do the following:
Locate the “SMTutorial_TransitionList.csv” file and open it in Excel.
On the Skyline Edit menu, choose Insert and click Transition List.
Skyline will show the Insert form, which may start out looking like this:
If it does, you can change it to accept small molecule fields by doing the following:
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Click the Small molecules option at the bottom of the form.
The form should now look like this:
In the transition list spreadsheet, you should find the following values:
Precursor Precursor Precursor Precursor Product Product
Molecule List Name Precursor Name Precursor Formula Adduct Charge RT CE m/z Charge Label
Amino Acid Methionine C5H11NO2S [M+H] 1 2.5 15 104.07 1
Amino Acid Methionine C5H8H'3NO2S [M+H] 1 2.5 15 107.09 1 heavy
Amino Acid Isoleucine C6H13NO2 [M+H] 1 2.9 15 86.096 1
Amino Acid Leucine C6H13NO2 [M+H] 1 3 15 86.096 1
Amino Acid Leucine C6H10H'3NO2 [M+H] 1 3 15 89.1 1 heavy
Amino Acid Phenylalanine C9H11NO2 [M+H] 1 3.1 15 120.08 1
Amino Acid Phenylalanine C9H11NO2 [M6C13+H] 1 3.1 15 126.11 1 heavy
Amino Acid Arginine C6H14N4O2 [M+H] 1 2.01 15 116.07 1
Amino Acid Arginine C1C'5H14N4O2 [M+H] 1 2.01 15 121.11 1 heavy
Amino Acid Ornithine C5H12N2O2 [M+H] 1 0.85 15 70.07 1
Amino Acid Ornithine C5H12N2O2 [M+H] 1 0.85 15 116.07 1
Amino Acid Ornithine C5H10H'2N2O2 [M+H] 1 0.85 15 72.07 1 heavy
Amino Acid Ornithine C5H10H'2N2O2 [M+H] 1 0.85 15 118.07 1 heavy
Organic Acid creatine C4H9N3O2 [M+H] 1 1.1 15 90.06 1
Organic Acid creatine C4H6H'3N3O2 [M+H] 1 1.1 15 93.06 1 heavy
5'-methylthioadenosine MTA C11H15N5O3S [M+H] 1 3.4 15 136.1 1
5'-methylthioadenosine MTA C11H12H'3N5O3S [M+H] 1 3.4 15 136.1 1 heavy
S-adenosyl methionine SAM C15H22N6O5S [M+H] 1 2.9 15 250.11 1
S-Adenosyl homocysteine SAH C14H20N6O5S [M+H] 1 2.9 15 136.08 1
Polyamine Spermidine C7H19N3[M+H] 1 3.3 15 129.15 1
Polyamine Spermine C10H26N4 [M+H] 1 3.5 15 112.112 1
Further notes on ion formulas and adduct descriptions
In this example, there are some light-heavy isotope label pairs, e.g. Methionine and d3-Methionine. In
the transition list they are described as C5H11NO2S [M+H] and C5H8H'3NO2S [M+H], but using the
language of adduct descriptions they could also have been described as C5H11NO2S [M+H] and
C5H11NO2S [M3H2+H]. You can see an example of this in the Phenylalanine heavy-light pair: the
formulas are the same but the heavy adduct description specifies that 6 carbons are replaced with C13.
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Note that the Adduct column is optional: as in the example of Spermidine, the adduct can be given as
part of the ion formula. Note also that the Precursor Charge column is not actually needed: the charge
states can be inferred from the adduct descriptions. The label column is also not strictly needed: the
heavy label can be inferred from the precursor ion formula and/or adduct.
You can see that there are some extra column headers in the Insert form, and the column order is not
the same in the form as in the spreadsheet. Both issues are easy to correct:
Click the Columns button and uncheck the columns that do not appear in the spreadsheet.
This should result in a column picking menu like the one shown below:
Next do the following to reorder the columns in the Insert form:
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Click and drag each column header you want to move to the order matching the spreadsheet.
Once you have selected and arranged your columns, the insert form should now appear as
shown below:
To add the transitions specified in the spreadsheet, do the following:
Select the contents of the spreadsheet, excluding the first row containing the headers.
Click the Copy button on the toolbar.
Switch back to Skyline.
Press Ctrl-V on your keyboard to paste.
Click the Check for Errors button.
NOTE: If you accidentally copied the header row or got the column order wrong, then you will see an
error at this point. Otherwise, the Insert form should look like this:
Click the Insert button.
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Your Skyline window should now look like:
At this point, either a native instrument method, precursor isolation list (for PRM) or transition list (for
SRM) can be exported. For more details on how to perform this step, please see the Targeted Method
Editing, Existing and Quantitative Experiments or Targeted MS/MS (PRM) tutorial.
Importing mass spectrometer runs
In this tutorial, you will simply import raw data from a Waters Xevo TQS instrument acquired using a
MassLynx instrument method exported by Skyline. To do this now, perform the following steps.
On the File menu, click Save. (Ctrl-S)
Save this document as “Amino Acid [Link]” in the tutorial folder you created.
On the File menu, select Import and click on Results.
In the Import Results form, choose to import single-injection replicates. For best performance,
be sure to select “Many” in the “Files to import simultaneously” control at the bottom of the
form. Now click the OK button.
Select all 18 raw data folders in the tutorial folder by clicking the first listed and then holding
down the Shift key and clicking the last.
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The Import Results Files form should look like:
Click the Open button.
Click the Do not remove button when asked about removing the common prefix.
The files are a metabolite extract of cancer cell lines under specific amino acid deprivation conditions,
where cells were deprived of either the amino acid Methionine, or Arginine, or both, for a period of 3
hours versus control (all amino acids).1
Filenames and Conditions:
ID15739_01_WAA263_3976_020415 – double blank
ID15740_01_WAA263_3976_020415 – Extraction Blank (contains SIL standards)
ID15740_02_WAA263_3976_020415 – Extraction Blank (contains SIL standards)
ID15740_04_WAA263_3976_020415 – Extraction Blank (contains SIL standards)
ID15655_01_WAA263_3976_020415 – All AA Sample 1
ID15656_01_WAA263_3976_020415 – All AA Sample 2
ID15657_01_WAA263_3976_020415 – All AA Sample 3
ID15658_01_WAA263_3976_020415 – Minus Met Sample 1
ID15659_01_WAA263_3976_020415 – Minus Met Sample 2
ID15660_01_WAA263_3976_020415 – Minus Met Sample 3
ID15661_01_WAA263_3976_020415 – Minus Arg Sample 1
ID15662_01_WAA263_3976_020415 – Minus Arg Sample 2
ID15663_01_WAA263_3976_020415 – Minus Arg Sample 3
ID15664_01_WAA263_3976_020415 – Minus Arg, Minus Met Sample 1
ID15665_01_WAA263_3976_020415 – Minus Arg, Minus Met Sample 2
ID15666_01_WAA263_3976_020415 – Minus Arg, Minus Met Sample 3
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ID15741_01_WAA263_3976_020415 – Pooled QC Sample 1
ID15741_02_WAA263_3976_020415 – Pooled QC Sample 2
The files should import within a matter of seconds, leaving your Skyline window looking like:
To take advantage of the Skyline summary graphs for viewing individual targets, do the following:
On the View menu, choose Peak Areas and click Replicate Comparison.
On the View menu, choose Retention Times and click Peak Areas.
Click and drag these views to dock them above the chromatogram graphs.
Select the first target “Methionine” in the Targets view.
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The Skyline window should now look something like this:
Conclusion
In this tutorial, you have learned how to create a Skyline document that targets small molecules
specified as precursor ion chemical formulas and adducts, and product ion m/z values. You imported a
multi-replicate data set collected by a metabolomics researcher, and saw how many existing Skyline
features created initially for targeted proteomics use can now be applied to small molecule data. Small
molecule support is still a relatively new feature area for Skyline. As such, you can expect it to continue
improving rapidly.
Bibliography
1. Tang, X. et al. Comprehensive Profiling of Amino Acid Response Uncovers Unique Methionine-
Deprived Response Dependent on Intact Creatine Biosynthesis. PLoS Genet 11, e1005158 (2015).