The identity evidence on a certificate of analysis is a mass measurement. How to read a peptide sequence explains how to calculate the theoretical mass from the sequence. This article starts where that one stops: at the spectrum itself.
What the x-axis means
A mass spectrometer does not measure mass but the ratio of mass to charge: m/z. That is why the x-axis reads m/z and not dalton. For a singly charged particle the two are almost the same; for a particle with three charges they differ by a factor of three. So every peak raises two questions: which mass, and which charge?
Two ways to ionise
A molecule first has to pick up a charge and reach the gas phase without falling apart. For large biomolecules that was the obstacle for a long time. In 2002 half of the Nobel Prize in Chemistry went to John B. Fenn and Koichi Tanaka “for their development of soft desorption ionisation methods for mass spectrometric analyses of biological macromolecules”; the other half went to Kurt Wüthrich for work with nuclear magnetic resonance. Fenn worked with electrospray ionisation (ESI), in which a solution is sprayed in an electric field; Tanaka showed that laser desorption works too, the approach used in MALDI.
Which of the two a certificate reports shapes what the spectrum looks like. With electrospray, a peptide takes up one or more protons and you usually see a series of peaks from the same molecule.
Ladders of charges
A protonated peptide is written as [M+zH]z+: the neutral mass M plus z protons, spread over z charges. PubChem gives the proton (the hydrogen cation) a monoisotopic mass of 1.00783. With that you can calculate the m/z of every charge state. Take BPC-157, with a monoisotopic mass of 1418.704 according to PubChem:
| Ion | Calculation | m/z |
|---|---|---|
| [M+H]+ | (1418.704 + 1.008) / 1 | 1419.71 |
| [M+2H]2+ | (1418.704 + 2 × 1.008) / 2 | 710.36 |
| [M+3H]3+ | (1418.704 + 3 × 1.008) / 3 | 473.91 |
It works just as well in reverse: from a peak at m/z 710.36 with charge 2 it follows that M = 2 × 710.36 – 2 × 1.008 = 1418.70. Working back from m/z to the neutral mass is called deconvolution. Software does it, but two peaks and a calculator get you there as well.
Monoisotopic or average
PubChem gives two masses per compound. For BPC-157 that is 1418.70 monoisotopic and 1419.5 average. The monoisotopic mass belongs to the molecule in which every atom is the most common isotope; the average mass accounts for the natural distribution of all isotopes and is therefore slightly higher.
Which of the two a certificate reports should be stated on it. A difference of almost one unit is not a rounding error for a small peptide, and for a long chain the gap grows. So always compare like with like.
Isotope pattern
At sufficient resolution, a peptide peak is not a line but a small cluster. That is because part of the molecules contain a heavier isotope, carbon-13 above all. Each extra isotope makes the molecule about one mass unit heavier, and on the m/z axis that unit is divided by the charge. So the spacing between the peaks in the cluster is about 1 at charge 1, about 0.5 at charge 2 and about 0.33 at charge 3. That lets you read the charge straight off the pattern.
Peaks that do not belong to the peptide
Not every peak is an impurity. A peptide can also pick up a sodium or potassium ion instead of a proton. With the values from PubChem the shift can be named exactly. For BPC-157:
- [M+Na]+ at m/z 1441.69. Sodium weighs 22.990, so 21.98 more than the proton.
- [M+K]+ at m/z 1457.67. Potassium weighs 38.964, so 37.96 more than the proton.
Other shifts point to a chemical change in the peptide itself. Manning and colleagues (Pharmaceutical Research, 2010) describe the reactions behind them:
- Oxidation: one extra oxygen atom. PubChem gives the oxygen atom as 15.995, so about +16 per oxidised side chain.
- Deamidation: an amide group (NH2) becomes an acid group (OH). That difference is exactly the difference between water (18.0106) and ammonia (17.0265) in PubChem: +0.98, in practice about +1.
- Loss of water: -18.01, the mass of one water molecule according to PubChem.
- A missing amino acid: a shift downwards by the mass of that residue, which is the mass of the free amino acid minus one water molecule. For proline PubChem gives 115.063; minus 18.011 that is 97.05.
Where those by-products come from during synthesis is set out in How are peptides made?; how to keep them from forming in storage is in Storing peptides in the lab.
Two examples from practice
One name, two compounds. In this catalogue, TB-500 is the fragment Ac-LKKTETQ with a molecular weight of 889.0 according to PubChem. The complete protein thymosin beta-4 has a weight of about 4963 in PubChem. So a mass measurement settles at once which of the two is in the vial, even when the label uses the same name.
With or without DAC. CJC-1295 exists in two forms: with DAC, at a molecular weight of 3647.2, and without DAC, at 3367.9. A difference of almost 280 is impossible to miss in a spectrum.
In a blend, every component should have its own mass; that is the subject of Peptide blends in research.
A mass spectrum never stands alone on a certificate: purity comes from the chromatogram and identity from the spectrum. How the two together describe a batch is explained in What is a COA? and What does 99% purity mean? The certificates for our batches are on Lab results.
Sources
Accessed in November 2026.
- The Nobel Prize in Chemistry 2002 (Nobel Prize Outreach): John B. Fenn and Koichi Tanaka, with the citation on soft desorption ionisation methods, and Kurt Wüthrich for nuclear magnetic resonance
- Hydrogen cation (PubChem), CID 1038: monoisotopic mass 1.00783
- Sodium ion (PubChem), CID 923: 22.98977, and Potassium cation, CID 813: 38.96371
- Water (PubChem), CID 962: 18.01056, Ammonia, CID 222: 17.02655, and Atomic oxygen, CID 159832: 15.99491
- L-Proline (PubChem), CID 145742: 115.063
- BPC-157 (PubChem), CID 9941957: 1418.704 monoisotopic and 1419.5 average
- TB-500 (Ac-LKKTETQ) (PubChem), CID 62707662: 889.0, and Thymosin Beta 4, CID 45382195: about 4963
- CJC-1295 (with DAC) (PubChem), CID 91971820: 3647.2, and CJC-1295 without DAC, CID 56841945: 3367.9
- Manning M.C. et al. (2010), Stability of protein pharmaceuticals: an update, Pharmaceutical Research (PubMed), PMID 20143256: oxidation, deamidation and hydrolysis
This article is background information on laboratory research. It is not medical advice or instructions for use. All products are for research use only.
