A peptide mass spectrum rarely contains only protonated ions. Next to the expected [M+H]+ peak there is often a companion about 22 units higher and sometimes another about 38 units higher. These are sodium and potassium adducts, and they are one of the most common reasons a correct material appears at first glance to contain an unexpected species.
Reading them correctly is mostly arithmetic. This guide gives the exact mass offsets, shows how they change with charge state, and explains how to separate a harmless adduct from a real modification that happens to sit nearby.
Where the +22 and +38 Da offsets come from
In positive-mode ESI or MALDI, a peptide becomes charged by picking up a cation. A proton is the usual one, but sodium and potassium ions are present almost everywhere at trace levels: borosilicate glassware, buffer salts, reagents, and handling all contribute. When a metal ion carries the charge instead of a proton, the ion mass rises by the difference between the two cations.
| Ion | Mass added to M (Da) | Offset from [M+H]+ |
|---|---|---|
| [M+H]+ | 1.00728 | 0 |
| [M+NH4]+ | 18.03383 | +17.02655 |
| [M+Na]+ | 22.98922 | +21.98194 |
| [M+K]+ | 38.96316 | +37.95588 |
The nominal “+22” and “+38” labels therefore describe offsets from the protonated ion, not from the neutral mass. The ammonium adduct appears mainly when ammonium formate, ammonium acetate or ammonium bicarbonate is in the mobile phase or sample. Acidic side chains and the C-terminal carboxylate can also exchange a proton for sodium, so a ladder of [M+H]+, +22, +44 and +66 indicates multiple exchanges rather than several different impurities.
Adduct intensity is not a fixed property of a peptide. Sequences with several aspartate or glutamate residues tend to show stronger sodium forms, and the same material can give quite different adduct ratios on two days or two instruments. The relative height of an M+22 peak therefore says more about trace salt in the sample and the ion source settings than about the quality of the peptide itself.
Adduct spacing at higher charge states
In ESI, larger peptides appear as multiply charged ions, and adducts follow the same rule as everything else: the m/z offset equals the mass offset divided by the charge. A sodium adduct on a doubly charged ion sits about 11 m/z above [M+2H]2+, and on a triply charged ion about 7.3 m/z above it. Potassium gives roughly 19 m/z at 2+ and 12.7 m/z at 3+.
Deconvolution software, which converts a charge-state series into neutral masses, will usually report these adduct series as a second mass about 22 or 38 Da above the main one. A deconvoluted result listing M, M+22 and M+38 describes one compound with three kinds of charge carrier. See our explanation of deconvolution of multiply charged spectra for how those lists are generated.
Worked example: annotating a 1,200 Da peptide spectrum
Take a hypothetical peptide with a monoisotopic neutral mass of 1,200.600 Da. The ions a laboratory might see are:
| Ion | Expected m/z |
|---|---|
| [M+2H]2+ | 601.307 |
| [M+H+Na]2+ | 612.298 |
| [M+H+K]2+ | 620.285 |
| [M+H]+ | 1201.607 |
| [M+NH4]+ | 1218.634 |
| [M+Na]+ | 1223.589 |
| [M+K]+ | 1239.563 |
Each of these belongs to the same molecule. Once assigned, none should appear in a list of unexplained species. Isotope spacing confirms the charge state of each ion before any assignment is made, as described in isotope patterns in peptide spectra. Sodium has a single stable isotope, so a sodium adduct keeps the ordinary carbon-13 envelope; potassium adducts carry an extra contribution two units up from potassium-41, which makes up about 6.7 percent of natural potassium.
When a potassium adduct imitates oxidation
The most useful trap to know involves combinations. The difference between potassium and sodium adducts is 15.974 Da, and a single oxygen atom adds 15.995 Da. A sodiated, oxidized peptide, [M+O+Na]+, therefore lands only about 0.021 Da away from [M+K]+. In the example above that is 1239.584 versus 1239.563, a gap of roughly 17 ppm.
At high resolution and good calibration the two are distinguishable by mass alone; the principles are covered in mass accuracy in ppm. On a nominal-mass instrument they are not, and chromatography becomes the deciding evidence. An adduct forms in the ion source, so its extracted ion chromatogram has exactly the same retention time and shape as the protonated main peak. A genuinely oxidized peptide, such as a methionine sulfoxide variant, is a different molecule and typically elutes at a slightly different time on reverse-phase HPLC.
Other adducts that mislead: TFA, chloride and matrix ions
- Trifluoroacetic acid. Peptides isolated as TFA salts may show an ion 113.993 Da above the corresponding protonated or deprotonated species. This reflects the counter-ion, not a covalent change; see TFA versus acetate counter-ions.
- Chloride in negative mode. [M+Cl]− sits about 35.98 Da above [M−H]−, with a chlorine-37 partner two units higher at roughly one third the intensity.
- MALDI matrix adducts. Matrix molecules can attach to analyte ions, producing peaks at offsets equal to the matrix mass or fragments of it.
What adducts mean for identity and purity results
Adducts do not reduce purity. HPLC purity is measured on separated species in solution, and adduct ions are formed afterwards, inside the mass spectrometer. What they do affect is signal: when a large share of the ion current goes to sodium or potassium forms, the [M+H]+ peak weakens, and quantitative LC-MS methods must either sum all adduct forms or control them. Laboratories reduce adducts with plastic rather than glass containers, fresh high-purity solvents, desalting before direct infusion, and LC separation that sends inorganic salts through with the solvent front.
For a purchaser, the practical rule is that an identity report should annotate adduct peaks rather than leave them unexplained. A spectrum showing M+22 and M+38 labeled as sodium and potassium adducts is consistent with a correct material. On our side, each product in the shop comes with an independent reverse-phase HPLC result, and a sealed vial is paired with the result published for it through its crimp and cap color, with no lot code involved.
Frequently asked questions
Is a peak at M+22 an impurity?
Almost always not. It is the sodium adduct of the main compound, formed during ionization, and it co-elutes exactly with the protonated ion.
Why is the sodium offset 21.98 Da and not 23?
Sodium replaces the proton as the charge carrier, so the offset from [M+H]+ is the sodium ion mass minus the proton mass.
How can a potassium adduct be told apart from oxidation?
By accurate mass, where the gap is about 0.021 Da, or by retention time, since an oxidized variant usually separates chromatographically while an adduct does not.
Do adducts affect HPLC purity values?
No. They form in the ion source after separation and have no effect on UV peak areas.
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