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Why a Peptide Mass Spectrum Has a Cluster of Peaks: Monoisotopic vs Average Mass Explained
Open a peptide mass spectrum and you will see a small forest of peaks where you might have expected a single spike. It is not noise. It is the fingerprint of carbon, hydrogen and nitrogen as they occur in nature, and once you know how to read it, you can check a peptide’s identity in seconds.
Key takeaways
- Most elements come in more than one isotope; carbon is about 1.1 percent carbon-13.
- Monoisotopic mass uses the lightest isotope of each element; average mass uses natural abundance.
- For small peptides, the first peak in the cluster is the monoisotopic one.
- Peak spacing equals one divided by the charge, so spacing reveals charge.
- Compare the correct kind of mass to avoid false mismatches.
Two kinds of mass
Every element has a main isotope and a few heavier ones. Carbon is mostly carbon-12, with about 1.1 percent carbon-13. A peptide with many carbons will therefore contain a few heavy atoms in a certain fraction of its molecules. The lightest possible version of the molecule, made only from the most common isotopes, has the monoisotopic mass. The weighted average over all isotopes is the average mass. Chemists often quote the average, the way a chemical catalog does, while high-resolution mass spectrometers see the monoisotopic peak.
A worked example
- Met-enkephalin, Tyr-Gly-Gly-Phe-Met, has the formula C27H35N5O7S.
- Monoisotopic mass: about 573.226.
- Average mass: about 573.7, the number you get by adding approximate residue masses, as in our sequence reading guide.
- In positive mode, the protonated ion [M+H]+ appears at about 574.233, because a proton adds 1.007.
- The next peak, M+1, sits about one mass unit higher and has an intensity that depends on how many carbons and other heavy-isotope-bearing atoms are present.
The cluster grows with size
A small peptide with 20 or so carbons has a strong monoisotopic peak and a smaller M+1. For a 30-carbon peptide, M+1 becomes about a third as intense. For a peptide of 2,000 daltons, the M+1 peak is roughly as intense as the monoisotopic one, and for larger molecules it takes over. Sulfur adds a small M+2 peak because of sulfur-34, which makes cysteine- or methionine-containing peptides slightly different in pattern.
Spacing and charge
Peptides in electrospray often carry more than one charge. The cluster peaks are one mass unit apart, but the instrument plots mass divided by charge. So a doubly charged ion has peaks half a unit apart, a triply charged one a third of a unit apart, and so on. Reading the spacing tells you the charge, as described further in our charge states post. That lets you compute the neutral mass of the peptide.
Common mistakes
- Comparing a monoisotopic measurement to an average mass from a calculator.
- Forgetting to subtract the proton mass for [M+H]+.
- Calling the tallest peak monoisotopic when it is actually M+1 in a larger peptide.
- Mistaking sodium or potassium adducts, which add 22 and 38, for impurities, as in our impurities post.
The bottom line
Isotope patterns look complicated, but they are simple arithmetic and a good check on any lab report. Learn the monoisotopic and average masses for your sequence, and the spectrum becomes readable. See HPLC versus mass spectrometry and our lab reports.
Related reading
- How to read a peptide sequence
- MS/MS sequencing
- HPLC versus mass spectrometry
- Browse the full Research Library
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For laboratory research use only. Not for human or veterinary use, and not intended to diagnose, treat, cure or prevent any condition. This article is educational and is not medical advice.