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Mass Spectrometry Explained: Confirming Peptide Identity
How mass spectrometry weighs a peptide, how to read charge states and adducts on a spectrum, and the difference between average and monoisotopic mass.
Bench card
- Mass spectrometry measures mass-to-charge ratio (m/z), not mass directly.
- Electrospray gives several charge states; MALDI mostly gives singly charged ions.
- Average and monoisotopic masses differ; check which one the COA reports.
- Sodium and potassium adducts appear as +22 and +38 Da peaks and are not impurities.
- Mass confirms composition, not sequence order. MS/MS fragmentation confirms sequence.
Mass spectrometry (MS) is the standard identity test for peptides. It answers the question “is the molecule in this vial the one on the label?” by measuring its mass very precisely.
The basic idea
A mass spectrometer turns molecules into ions, separates them by mass-to-charge ratio (m/z), and counts them. The spectrum plots signal intensity against m/z. For a peptide carrying one extra proton, m/z is the molecular mass plus about 1.007 Da.
Ionization: ESI and MALDI
| Method | How it works | What you see |
|---|---|---|
| Electrospray ionization (ESI) | A solution is sprayed through a charged needle into fine droplets that evaporate, leaving charged ions | Several charge states ([M+H]+, [M+2H]2+, …) |
| Matrix-assisted laser desorption/ionization (MALDI) | The sample is co-crystallized with a matrix and fired at with a laser | Mostly singly charged [M+H]+ |
Fenn and colleagues described electrospray for large biomolecules in 1989, work recognized in the 2002 Nobel Prize in Chemistry; Karas and Hillenkamp described MALDI of proteins above 10,000 Da in 1988.
Reading charge states
For an ion carrying z extra protons, m/z = (M + z × 1.007) ÷ z. For a peptide of mass 1,419.5 Da:
| Charge | Expected m/z |
|---|---|
| 1+ | about 1,420.5 |
| 2+ | about 710.8 |
| 3+ | about 474.2 |
Software can deconvolute a series of charge states into a single neutral mass, which is what many COAs report.
Average vs monoisotopic mass
Elements have isotopes; carbon is about 1.1% 13C. The monoisotopic mass uses only the most abundant isotope of each element. The average mass uses standard atomic weights, which reflect natural isotope abundance. For larger peptides the most intense peak is not the monoisotopic one, and many reports quote average mass. Check which convention a COA uses before comparing numbers; the difference grows with size, roughly 1 Da per 1,800 Da.
Peaks that are not impurities
- +22 Da: sodium adduct ([M+Na]+).
- +38 Da: potassium adduct ([M+K]+).
- Isotope peaks: a cluster spaced 1/z apart.
Peaks that may be impurities
- +16 Da: oxidation, often Met to Met sulfoxide.
- +1 Da: deamidation (Asn to Asp); hard to see without high resolution.
- Minus one residue mass: a deletion sequence from synthesis.
- +96 Da: a trifluoroacetyl adduct or modification, depending on context.
References
- Fenn JB, Mann M, Meng CK, Wong SF, Whitehouse CM. Electrospray ionization for mass spectrometry of large biomolecules. Science. 1989;246(4926):64–71. DOI
- Karas M, Hillenkamp F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem. 1988;60(20):2299–2301. DOI
- Aebersold R, Mann M. Mass spectrometry-based proteomics. Nature. 2003;422(6928):198–207. DOI
- Meija J, Coplen TB, Berglund M, et al. Atomic weights of the elements 2013 (IUPAC Technical Report). Pure Appl Chem. 2016;88(3):265–291. DOI
- NIST. Atomic Weights and Isotopic Compositions for All Elements. physics.nist.gov
- IUPAC. Compendium of Chemical Terminology (the "Gold Book"). goldbook.iupac.org