Guide · 7 min read
Mass spectrometry explained: verifying a peptide's identity
HPLC tells you how pure a sample is. Mass spectrometry tells you what it is. If you only check one number on a peptide COA, check the mass. Here's how to interpret it, including the parts that trip people up.
What a mass spectrometer measures
A mass spectrometer turns molecules into charged ions, separates them in an electric or magnetic field, and records their mass-to-charge ratio (m/z). Peptide COAs typically use one of two techniques:
- ESI-MS (electrospray ionization), often coupled to HPLC as LC-MS. It gently adds protons to the peptide, often several at once, so you see a series of peaks.
- MALDI-TOF, which usually produces singly-charged ions, so the main peak sits close to the peptide's mass plus one proton.
Theoretical mass: what you compare against
Every peptide has a molecular formula, and from that formula you can calculate an exact theoretical mass. There are two versions:
- Monoisotopic mass: calculated with the lightest common isotope of each element (¹²C, ¹H, ¹⁴N, ¹⁶O, ³²S). High-resolution instruments report this.
- Average molecular weight: uses the natural isotope mix. It's the number on most product pages, and it's what deconvoluted low-resolution spectra land close to.
| Compound | Monoisotopic (Da) | Average (Da) | Gap |
|---|---|---|---|
| Epitalon | 390.14 | 390.35 | 0.2 |
| BPC-157 | 1,418.70 | 1,419.54 | 0.8 |
| CJC-1295 (no DAC) | 3,365.89 | 3,367.90 | 2.0 |
| Tirzepatide | 4,810.52 | 4,813.45 | 2.9 |
That's why a good COA states which value it reports, and why the COA checker compares against both.
Charge states: why you see "1123.6" for a 3368 Da peptide
In ESI, a peptide of mass M picking up n protons appears at:
m/z = (M + n × 1.00728) ÷ n
For Mod GRF (1-29) (M ≈ 3367.9 Da): 3+ → 1123.6, 4+ → 843.0, 5+ → 674.6. The lab's software combines these into one neutral mass through a process called deconvolution. A COA that shows only one m/z value is fine as long as it matches one of these charge states. The checker tests charge states from 1+ to 8+ automatically.
Common offsets and what they mean
| Offset from expected | Likely cause | Concern level |
|---|---|---|
| +1.007 | [M+H]+: the lab reported the protonated ion | None |
| +21.98 | Sodium adduct [M+Na]+ | Low |
| +37.96 | Potassium adduct [M+K]+ | Low |
| +15.99 / +31.99 | Oxidation (methionine, tryptophan) | Moderate if it's the main peak |
| +2.02 | Reduced disulfide (open ring) | Moderate for cyclic peptides |
| −18.01 | Water loss (e.g. aspartimide or cyclization) | Moderate |
| +42.01 | Extra acetyl group (e.g. an N-acetylated variant) | Check the product name |
| Matches another peptide | Mislabeled product or a copied COA | High |
How close is close enough?
For typical COA-grade instruments, a match within ±1 Da (or about 0.03% for peptides above about 3 kDa) is a reasonable standard, and it's what this site's checker uses. High-resolution instruments (Orbitrap, Q-TOF) can do far better, matching to within a few parts per million. If a lab reports a value to four decimal places, it should be correspondingly close to the monoisotopic mass.
Look-alike pairs to watch
- Melanotan II (1024.2) vs PT-141 (1025.2): only about 1 Da apart.
- Sermorelin (3357.9) vs Mod GRF 1-29 (3367.9): about 10 Da apart.
- CJC-1295 no DAC (3367.9) vs with DAC (3647.2).
- TB-500 fragment (889.0) vs full Thymosin Beta-4 (4963).
- Tirzepatide (4813.5) vs Retatrutide (4731.3).
Not sure if your COA's mass matches? The checker converts m/z, spots adducts, and tells you if the mass belongs to a different peptide.
Check the mass now →Sources
- PubChem (U.S. National Library of Medicine): Reference formulas and masses.
- CIAAW: Standard atomic weights: Basis for average molecular weights.
- NIST CODATA: proton mass: Used to convert m/z charge states to neutral mass.
- PeptideCOACheck methodology: The exact rules the checker applies.
Last updated . Written and maintained under our editorial policy. Found an error? See corrections.
Frequently asked questions
How does mass spec verify a peptide?
Mass spectrometry measures the mass of the molecules in a sample. If the observed mass matches the peptide's theoretical molecular weight (calculated from its formula) within a small tolerance, that's strong evidence the sample contains the right molecule.
How close should the measured mass be?
For most research peptides, within about ±1 Da of the theoretical average or monoisotopic mass. For large peptides over about 3,000 Da, a tolerance of about 0.03% (roughly 1–1.5 Da) is reasonable for typical instruments. High-resolution instruments can match to a few parts per million.
Why is the mass on my COA much smaller than the molecular weight?
It is probably an m/z value for a multiply-charged ion. In electrospray MS, a peptide carrying n extra protons appears at (M + n × 1.007) / n. For example, a 3,367.9 Da peptide carrying 3 protons appears near 1,123.6 m/z.
What is the difference between average and monoisotopic mass?
Monoisotopic mass uses only the most common isotope of each element (carbon-12, hydrogen-1 and so on). Average mass uses the natural isotope mix. The average is always slightly higher, by less than 1 Da for small peptides and 2–3 Da for 4–5 kDa peptides.
Educational content only. Not medical advice. Research peptides are sold for laboratory research purposes only.