Reading a Peptide Mass Spectrum: ESI, MALDI, Charge States and Isotopes

In plain English

A mass spectrometer is a very precise scale for molecules. It cannot weigh a molecule directly. First it gives the molecule an electric charge (ionisation), then it measures how the charged molecule moves in an electric or magnetic field. The machine reports mass divided by charge, written m/z ^{[3, 4]}.

For peptides (short chains of amino acids), this is the main way to check identity. If a peptide should weigh 1,045.53 daltons (Da, the unit used for molecular mass) and the spectrum shows that mass, the identity is supported. If it shows something else, the vial may hold a different molecule. In one study of a research peptide bought from five manufacturers, one product turned out to be a completely different peptide ^[7].

Mass spectrometry confirms mass. On its own, it does not measure purity or the amount of peptide in a vial.

The short version

• The spectrum shows mass ÷ charge, not mass.
• One peptide can appear as several peaks with different charges.
• Isotopes make each peak a small cluster.
• Identity needs mass spectrometry; purity needs a separating method such as HPLC.

How peptides become ions: ESI and MALDI

A peptide must carry a charge and be in the gas phase before it can be measured. Two “soft” ionisation methods do this without breaking the peptide apart.

Electrospray was shown to give “coherent sequences of peaks” in which neighbouring ions differ by one charge, which is the pattern seen for peptides and proteins today ^[1]. Laser desorption with a matrix was shown in 1988 to ionise intact proteins above 10,000 Da ^[2].

What the axes mean

The x-axis is m/z: the mass of the ion divided by its number of charges. The y-axis is relative intensity, scaled so the tallest peak is 100%. Intensity reflects how well a species ionised and was detected, so peak height on a mass spectrum is not a reliable measure of how much of each species is present. That is one reason purity is measured by HPLC rather than MS ^{[3, 9]}.

Charge states: one peptide, several peaks

In positive-mode ESI, a peptide picks up protons (H⁺, mass 1.00728 Da). An ion with z extra protons appears at:

m/z = (M + z × 1.00728) ÷ z

where M is the neutral monoisotopic mass.

Worked example: angiotensin II

Angiotensin II (sequence DRVYIHPF) is a common calibration peptide. Its formula is C₅₀H₇₁N₁₃O₁₂, with a monoisotopic mass of 1045.5345 Da and an average molecular weight of 1046.2 g/mol ^[8].

An ESI spectrum of this peptide would typically show the 2+ ion strongly, with smaller 1+ and 3+ peaks. A MALDI spectrum would be dominated by the 1+ ion.

Working out the charge from two neighbouring peaks

If two peaks belong to the same peptide and differ by one charge, the charge of the higher-m/z peak is:

z = (lower m/z − 1.00728) ÷ (higher m/z − lower m/z)

Using 349.52 and 523.77: (349.52 − 1.00728) ÷ (523.77 − 349.52) = 348.51 ÷ 174.25 = 2.0. The 523.77 peak is therefore 2+, and the neutral mass is 2 × (523.77 − 1.00728) = 1045.53 Da, matching the theoretical value ^[8]. Software performs this “deconvolution” automatically, but the arithmetic is the same.

Isotopes: why each peak is a cluster

Carbon in nature is about 1.1% carbon-13. A peptide with 50 carbon atoms therefore has a good chance of containing at least one ¹³C atom, which adds about 1.003 Da. Each ion appears as an isotope envelope: a main peak followed by smaller peaks.

• Monoisotopic peak. The first peak in the cluster, containing only the lightest isotopes (¹²C, ¹H, ¹⁴N, ¹⁶O). High-resolution instruments report this value ^[3].
• Average mass. The weighted mean over all isotopes. This is the “molecular weight” listed in databases and on certificates (1046.2 for angiotensin II, versus 1045.53 monoisotopic) ^[8].
• Spacing reveals charge. Isotope peaks are about 1.003 apart for a 1+ ion, 0.50 apart for 2+, and 0.33 apart for 3+. On a high-resolution spectrum, the spacing alone gives the charge.

As peptides get larger, more of the molecules carry heavy isotopes, and the monoisotopic peak becomes a smaller part of the cluster. Comparing a monoisotopic measurement with an average theoretical mass (or the reverse) is a common source of an apparent “1 Da error”.

Adducts and contaminants

Not every peak is the target peptide or an impurity from synthesis. Common extra peaks include:

Sodium and potassium adducts, polymer contaminants and solvent clusters are well-documented background signals in ESI and MALDI ^[5]. Trifluoroacetic acid (TFA), common in peptide HPLC and as a counter-ion, can suppress the ESI signal through ion pairing and surface-tension effects ^[10]. See Peptide Counter-Ions: TFA and Acetate and Common Peptide Impurities.

Tandem MS: confirming the sequence

Matching the intact mass shows that the formula is plausible, but different sequences can share the same mass. Tandem mass spectrometry (MS/MS) breaks the selected ion into fragments. Breaks along the backbone produce b ions (containing the N-terminus) and y ions (containing the C-terminus). The mass differences between consecutive fragments correspond to individual amino acid residues, so the sequence can be read from the spectrum ^[3]. Leucine and isoleucine have identical masses and are not distinguished by standard fragmentation.

Reading the MS section of a COA

A useful mass spectrometry result on a Certificate of Analysis states:

1. The method (ESI or MALDI; LC-MS or direct infusion).
2. Observed and theoretical mass, with a note on whether each is monoisotopic or average.
3. The ion used (for example [M+2H]²⁺ at m/z 523.77) or a deconvoluted neutral mass.
4. An attached spectrum with labelled peaks.
5. An acceptance criterion, such as agreement within a stated tolerance.

International guidance on specifications for biotechnological products lists identity testing as a core part of a specification ^[9]. For how MS fits with HPLC purity, see HPLC, LC-MS and UV-Vis and How to Read a Peptide COA.

What this means in Australia

• Identity testing does not change regulatory status. A mass spectrum confirms what a molecule is. It does not enter a product on the Australian Register of Therapeutic Goods (ARTG), and unapproved peptide products have not been assessed by the TGA ^[11].
• Scheduling depends on the substance. Several peptides are named in the current Poisons Standard (October 2026), some in Schedule 4 (prescription-only) and Appendix D, clause 5 (possession without authority is illegal) ^[12]. Confirming identity by MS is how a laboratory knows which substance it actually holds.
• Labels don’t change status. The TGA has stated that a “research use only” disclaimer does not make supply lawful, change regulatory status, permit importation, or remove advertising or supply obligations ^[11].
• Accredited testing. In Australia, NATA accredits testing laboratories to ISO/IEC 17025, which is a useful signal when judging who produced a mass spectrometry report ^[13].

This is general information, not legal advice. Laws change; check the current Poisons Standard and TGA guidance.

Frequently asked questions

What does m/z mean in mass spectrometry?

m/z is the mass of an ion divided by its charge. A peptide carrying two protons appears at roughly half its mass, so the same molecule can produce several peaks ^[1].

What is the difference between ESI and MALDI?

ESI sprays a solution at high voltage and usually produces multiply charged ions, which suits LC-MS. MALDI uses a laser and a matrix and mostly produces singly charged ions ^{[1, 2, 4]}.

Why is the measured mass 1 Da different from the expected mass?

The usual cause is comparing a monoisotopic value with an average mass, or picking the wrong peak in the isotope cluster. Checking which mass type each number represents usually resolves it ^[8].

Can mass spectrometry measure peptide purity?

Not reliably on its own. Ionisation efficiency differs between molecules, so peak heights don’t reflect amounts. Purity is measured by a separating method such as HPLC, often combined with MS as LC-MS ^{[3, 9]}.

What is an [M+Na]⁺ peak?

It is the peptide carrying a sodium ion instead of a proton. It appears about 22 units above [M+H]⁺ and is a common background signal rather than an impurity in the peptide itself ^[5].

References

1. 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:10.1126/science.2675315. PMID: 2675315
2. Karas M, Hillenkamp F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem. 1988;60(20):2299–2301. doi:10.1021/ac00171a028. PMID: 3239801
3. Steen H, Mann M. The ABC’s (and XYZ’s) of peptide sequencing. Nat Rev Mol Cell Biol. 2004;5(9):699–711. doi:10.1038/nrm1468. PMID: 15340378
4. Domon B, Aebersold R. Mass spectrometry and protein analysis. Science. 2006;312(5771):212–217. doi:10.1126/science.1124619. PMID: 16614208
5. Keller BO, Sui J, Young AB, Whittal RM. Interferences and contaminants encountered in modern mass spectrometry. Anal Chim Acta. 2008;627(1):71–81. doi:10.1016/j.aca.2008.04.043. PMID: 18790129
6. D’Hondt M, Bracke N, Taevernier L, et al. Related impurities in peptide medicines. J Pharm Biomed Anal. 2014;101:2–30. doi:10.1016/j.jpba.2014.06.012. PMID: 25044089
7. De Spiegeleer B, Vergote V, Pezeshki A, Peremans K, Burvenich C. Impurity profiling quality control testing of synthetic peptides using liquid chromatography-photodiode array-fluorescence and liquid chromatography-electrospray ionization-mass spectrometry: the obestatin case. Anal Biochem. 2008;376(2):229–234. doi:10.1016/j.ab.2008.02.014. PMID: 18342612
8. National Center for Biotechnology Information. PubChem Compound Summary for CID 172198, Angiotensin II. pubchem.ncbi.nlm.nih.gov/compound/172198
9. International Council for Harmonisation. ICH Q6B: Specifications: test procedures and acceptance criteria for biotechnological/biological products. database.ich.org
10. Apffel A, Fischer S, Goldberg G, Goodley PC, Kuhlmann FE. Enhanced sensitivity for peptide mapping with electrospray liquid chromatography-mass spectrometry in the presence of signal suppression due to trifluoroacetic acid-containing mobile phases. J Chromatogr A. 1995;712(1):177–190. doi:10.1016/0021-9673(95)00175-m. PMID: 8556150
11. Therapeutic Goods Administration. Understanding your responsibilities when importing, compounding and supplying unapproved peptide products. Safety advisory, 13 April 2026. tga.gov.au. Accessed 11 October 2026.
12. Therapeutic Goods (Poisons Standard—October 2026) Instrument 2026 (F2026L01327). Federal Register of Legislation. legislation.gov.au. Accessed 11 October 2026.
13. National Association of Testing Authorities, Australia (NATA). Accreditation programs, including ISO/IEC 17025 testing and calibration. nata.com.au. Accessed 11 October 2026.

Important information and disclaimer

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Regulatory status. Many peptides are scheduled substances under the Poisons Standard. Some are prescription-only (Schedule 4), and some may not be possessed without authority (Appendix D). The TGA has stated that a “research use only” label does not make supply lawful, change a product’s regulatory status, or remove advertising or supply obligations. State and territory laws may also apply. You are responsible for knowing and complying with all laws that apply to you. Regulatory information in this article may change after the review date.

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