Reading a mass spectrum on a peptide COA: m/z, charge states and adducts

The mass spectrum is the identity panel on a peptide certificate of analysis. It answers one question: does the molecule in the vial have the mass expected from its sequence? The plot can look cryptic at first, because peaks rarely sit at the molecular weight printed on the label. This guide explains why, and walks through a worked example.

Electrospray ionization

Most peptide COAs use electrospray ionization (ESI), often coupled to liquid chromatography (LC-MS). The sample in solution is sprayed through a charged needle, and the peptide leaves the droplets as ions carrying one or more extra protons. ESI is gentle, so the peptide usually stays intact.

m/z, not mass

A mass spectrometer measures the mass-to-charge ratio, m/z. A peptide of mass M that has picked up n protons appears at:

m/z = (M + n × 1.007) / n

[M+H]+: one proton, appears at M + 1.
[M+2H]2+: two protons, appears at about half the mass plus 1.
[M+3H]3+: three protons, about one third of the mass plus 1.

Small peptides show mostly the 1+ ion. Larger ones carry more charges, because they have more basic sites (Lys, Arg, His and the N-terminus). A 3,000 to 5,000 Da peptide often shows a series of 3+, 4+ and 5+ ions and little or no 1+ ion.

Adducts

Ions can also form with cations other than protons. The most common is sodium:

[M+Na]+ appears about 22 units above [M+H]+ (sodium 22.99 minus hydrogen 1.01).
[M+K]+ appears about 38 units above [M+H]+.
Mixed ions such as [M+H+Na]2+ appear about 11 units above the [M+2H]2+ peak.

Small adduct peaks are normal and come from glassware, buffers and salts. They confirm the same molecule rather than indicating an impurity.

Monoisotopic vs average mass

Product pages list the average molecular weight, which accounts for the natural mix of isotopes. A high-resolution spectrum resolves the isotope pattern, and its first peak is the monoisotopic mass (all 12C, 1H, 14N, 16O). For a 1.4 kDa peptide the two differ by nearly 1 unit, so do not expect them to match exactly.

Deconvolution

When a spectrum shows several charge states, software can combine them and calculate the neutral mass, a step called deconvolution. Many COAs report this deconvolved mass next to the theoretical value. Agreement within about 1 Da, or a few ppm on high-resolution instruments, is typical.

Worked example: BPC-157

BPC-157 (GEPPPGKPADDAGLV) has the formula C62H98N16O22, an average mass of about 1419.6 g/mol and a monoisotopic mass of about 1418.70 Da. Expected ions:

Ion	Calculation	Expected m/z
[M+H]+	1418.70 + 1.007	≈ 1419.7
[M+2H]2+	(1418.70 + 2.015) / 2	≈ 710.4
[M+3H]3+	(1418.70 + 3.022) / 3	≈ 473.9
[M+Na]+	1418.70 + 22.99	≈ 1441.7
[M+H+Na]2+	(1418.70 + 1.007 + 22.99) / 2	≈ 721.3

BPC-157 has only one lysine and a free N-terminus, so a typical spectrum is dominated by the 1+ and 2+ ions. If the main peaks fall at about 1419.7 and 710.4, the identity is consistent with BPC-157.

Common shifts to recognize

+16: oxidation, usually of methionine.
+42: acetylation.
−18: loss of water, sometimes seen as an in-source fragment.
+1 (deamidation): Asn or Gln converted to Asp or Glu.

For more on why masses shift, see salts, caps and oxidation. For the overall identity check, see how mass spectrometry confirms identity, and pair it with the HPLC purity panel.

All LA Peptides products are supplied for laboratory research use only and are not for human or veterinary use.

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All LA Peptides products are sold for laboratory research only. Not for human or veterinary use.

For laboratory research use only. Not for human or veterinary use. This guide summarizes published scientific literature for context. It is not medical advice and does not describe any product as suitable for human use.

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