precursor isotopic envelope

precursor isotopic envelope bobxiong  2022-08-31

Hi Skyline Team,

In Skyline Proteomics interface, theoretical isotopic precursors with m/z values lower than the monoisotopic precursor were displayed. My understanding is that the monoisotopic species is of the lowest m/z (I could be wrong though as I am not familiar with the various ways of determining the isotopic envelope). I attached a screenshot here. The Skyline zip file was uploaded to (file name

I was trying to understand this to help interpret a related Skyline analysis in Molecule interface.

Thanks a lot,

Bob Xiong, Ph.D.
Joinn Laboratories
Beijing, China
Email address:

Nick Shulman responded:  2022-08-31
In your screenshot, the spectrum that you are looking at does not match your peptide. In between each of the isotope peaks that are expected to be there, there is another peak observed. That spectrum belongs to a charge 4 molecule.

The monoistopic mass is the mass which you get by using the most abundant isotopic mass of all each the elements. The most abundant mass is not necessarily the lowest stable isotopic mass, but, yes, for Carbon, Hydrogen, Nitrogen, Oxygen and Sulfur, the most abundant isotope is also the isotope with the lowest mass.
There are elements such as Iron for which the most abundant isotope is not the isotope with the lowest mass.

When you have a heavy labeled peptide, some percentage of the atoms which are supposed to be heavy are not actually heavy. You can tell Skyline what the expected atomic percent enrichment for these heavy labeled amino acids is by going to:
Settings > Transition Settings > Full Scan
and doing something with the "Isotope labeling encrichment" setting.
When you look at the isotopic envelope for a heavy labeled peptide, it always looks like it does in your screenshot because some of the atoms which were supposed to be heavy were not actually heavy.
-- Nick
bobxiong responded:  2022-08-31
Hi Nick,

Thanks for your quick and insightful response. Your mentioning of "a charge 4 molecule" made me put on my thinking cap. Indeed, that charge-4 of 1216.6 m/z represents a dimer molecule in the sample (skyline zip uploaded to (file name

I was wrong stating that the 1216.6 m/z was "theoretic". It was actually derived from the scan data following deconvolution.

Out of curiosity, how did you know the 1216.6 m/z was a charge 4? it was not labeled as such in the displayed gragh. I knew it was charge 4 in my dimer analysis. But you were not aware of my dimer analysis yesterday :)

Thanks again,

Nick Shulman responded:  2022-08-31
You can always determine the charge state of a molecule by looking at the distance between the isotope peaks in the mass spectrum.
You can see four isotope peaks between 1217 and 1218 in that spectrum so it must be a charge 4 molecule.
-- Nick