Mass spectrometry for peptides: how identity is confirmed

In short: Mass spectrometry (MS) confirms a peptide’s identity by measuring the mass-to-charge ratio of its ions and comparing the result with the mass calculated from the expected sequence. Tandem MS adds sequence evidence from fragment ions. A mass match is strong identity evidence, but it is not a purity result and cannot separate isomers.

Identity is one of the core results on a peptide certificate of analysis (COA), and mass spectrometry is a central method behind it. The European Medicines Agency (EMA) describes MS as a powerful tool for working out the structure of peptides 1. This explainer covers what the instrument measures, how tandem MS reads a sequence, and where a mass match stops being informative. It pairs with our explainer on what an HPLC purity percentage means.

What does a mass spectrometer measure?

A mass spectrometer measures the mass-to-charge ratio (m/z) of ions, not the mass of neutral molecules. United States Pharmacopeia (USP) general chapter <736> describes MS as a technique for determining molecular mass and elemental composition and for elucidating structure 2.

Before a peptide can be measured, it has to become a gas-phase ion carrying one or more extra protons. The output, a mass spectrum, plots signal intensity against m/z. An ion with one extra proton appears at an m/z close to the peptide’s mass. An ion with three extra protons appears at roughly one third of that value.

How are peptides turned into ions?

Peptide MS relies on “soft” ionisation methods, which move large, fragile molecules into the gas phase intact 3. Two such methods are electrospray ionisation (ESI) and matrix-assisted laser desorption/ionisation (MALDI).

Electrospray ionisation. In 1989, Fenn and colleagues described ESI as a way of producing intact ions from large molecules in solution. Its spectra show a series of peaks from multiply charged ions, each differing from its neighbours by one charge 3. Because ESI starts from a solution, it is often coupled directly to high-performance liquid chromatography (HPLC), a combination known as LC-MS 4.

The solvent matters. In a 2020 review, Sikora et al. noted that trifluoroacetic acid (TFA), a common HPLC additive for peptides, is not recommended for LC-MS because it causes relatively high ion suppression and lowers sensitivity; volatile additives such as formic or acetic acid suit LC-MS better 4. TFA also ends up bound to many synthetic peptides, as covered in acetate vs TFA salts in synthetic peptides.

MALDI. MALDI embeds the sample in a matrix and uses a laser pulse, most often ultraviolet, to desorb and ionise it 5. Karas and Hillenkamp reported laser desorption ionisation of proteins above 10,000 daltons in 1988 6.

Illustration: electrospray ionisation turns peptides in solution into gas-phase ions.

How is a measured mass matched to the expected sequence?

The laboratory calculates the mass the correct peptide should have from its sequence, then checks that the observed ions agree within a stated tolerance.

Monoisotopic or average mass. Two kinds of calculated mass are used. The monoisotopic mass is built from the most abundant isotope of each element. The average mass, which is the familiar molecular weight, weights every isotope by how common it is. For L-leucine, PubChem lists a molecular weight of 131.17 g/mol and a monoisotopic mass of 131.0946 Da 7. A COA should say which one it reports, because the two values are not interchangeable.

Charge states. With ESI, one peptide usually appears as several peaks carrying different numbers of protons 3. Software converts that series of m/z values back to a single neutral mass, a step called deconvolution.

Mass accuracy. Agreement is expressed as a mass error, often in parts per million (ppm). Zubarev and Mann noted in 2007 that “mass accuracy” was used in at least three different senses in proteomics, and proposed recalibrating each dataset on internal standards 8. For medicines, the EMA’s synthetic peptide guideline asks for representative spectra with tables of theoretical and observed mass values 1.

Some changes to a peptide shift its mass clearly; others shift it slightly, or not at all.

Change to the peptide or ion	Monoisotopic mass difference (Da)	Visible as a mass change?	Source
Methionine oxidation (one added oxygen)	+15.9949	Yes	Unimod 9
Deamidation of asparagine or glutamine	+0.9840	Yes, as a small shift	Unimod 9
Sodium adduct (sodium in place of a proton on the ion)	+21.9819	Yes, but Unimod classes it as an artefact, not a change to the peptide	Unimod 9
One glycine residue missing (a deletion sequence)	-57.0215	Yes	Residue mass from Medzihradszky and Chalkley 10
Isoleucine in place of leucine	0	No: same formula, C6H13NO2	PubChem 7
Glutamine in place of lysine	0.0364	Only with high mass accuracy	PubChem 7; 10
An L-amino acid replaced by its D-form	0	No: the two forms share a formula	PubChem, L- and D-alanine 7

How does tandem mass spectrometry read a sequence?

Tandem mass spectrometry (MS/MS) selects one ion, breaks it apart and measures the pieces. Medzihradszky and Chalkley’s 2015 tutorial describes collision-induced dissociation (CID), in which the selected ion is energised by collisions, as the most common activation method 10.

Cleavage along the peptide backbone gives fragments that keep the charge on the N-terminal side (a, b and c ions) or on the C-terminal side (x, y and z ions), each numbered from its own end 10. Roepstorff and Fohlman proposed the first shared naming scheme for these ions in 1984 11.

The useful part is the spacing. The mass difference between neighbouring members of one ion series equals the mass of one amino acid residue, so a ladder of b or y ions spells out part of the sequence 10. The same tutorial sets out the limits. Leucine and isoleucine cannot be told apart this way. The 36 millidalton gap between glutamine and lysine is about 120 ppm at m/z 300 but only 25 ppm at m/z 1,500, so the right fragments have to be measured accurately 10.

The EMA guideline lists LC-MS/MS of the intact molecule as a technique for confirming amino acid sequence. For long peptides, where MS/MS data may be difficult, it points to peptide mapping, in which the peptide is cut into pieces that are analysed separately 1.

Illustration: tandem MS breaks a peptide along its backbone and measures the fragments.

What can mass spectrometry not tell you?

A correct mass shows that the expected molecule is present. It does not show how much of the sample that molecule makes up, or what else is there.

Purity. Signal size in a mass spectrum depends on how readily each species ionises, and additives can suppress ionisation 4. The EMA guideline treats purity as a separate specification test, and notes that the assay is usually based on the same chromatographic method as purity 1. Our explainer on HPLC purity covers that measurement.

Isomers. Molecules with the same formula give the same mass. A 2021 review by Lian et al. gave particular attention to structural isomers and stereoisomers, including peptide epimers (one amino acid in its D-form), among the challenges of characterising synthetic peptides by LC-MS 12. The EMA guideline names chiral gas chromatography with MS detection, or LC methods, for checking the D and L forms of amino acids 1.

Related impurities. A 2014 review by D’Hondt et al. catalogued impurities in synthetic peptide medicines, including deletion and insertion sequences, diastereomers, leftover protecting groups, oxidation products and unrelated peptides 13. Most differ in mass and show up in LC-MS; diastereomers do not. In a 2018 analytical study, Li et al. used LC with high-resolution MS to identify more than 65 structurally related impurities in a synthetic human C-peptide material, totalling 83.3 mg/g 14. For medicines, European Pharmacopoeia (Ph. Eur.) thresholds cited by the EMA call for peptide-related impurities to be reported above 0.1%, identified above 0.5% and qualified above 1.0% 1.

Low-level cross-contamination. In a 2008 laboratory study using human T-cell assays, Currier et al. traced false-positive responses to a library peptide that contained about 1% by weight of an unrelated cytomegalovirus peptide 15.

Counter-ions and water. An identity result says nothing about how much of a vial’s weight is counter-ion or water. The EMA guideline lists both as separate specification tests 1, and both affect net peptide content.

Why do guidelines ask for more than one identity test?

Because each method has blind spots, regulators ask for a combination. For medicines, the EMA guideline recommends at least two orthogonal (independent) identification methods. It names mass, relative retention time, LC-MS, peptide mapping, bioactivity, amino acid analysis and nuclear magnetic resonance (NMR) spectroscopy as options, and asks that the combination unambiguously confirm the sequence 1.

For a multi-component blend, the same reasoning applies to each component, as covered in how peptide blends are tested.

How should a mass spectrometry result on a COA be read?

A mass spectrometry identity result is easiest to check when it states five things:

• the expected mass, and whether it is monoisotopic or average
• the observed value, as m/z with charge states or as a deconvoluted neutral mass
• the difference between expected and observed, in daltons or ppm
• the ionisation method (ESI or MALDI) and whether the instrument was coupled to LC
• whether MS/MS or another orthogonal identity test was also performed.

A result that says only “conforms” without these values is harder to check independently.

Frequently asked questions

What does m/z mean on a peptide COA?

m/z is the mass-to-charge ratio of an ion, which is the quantity a mass spectrometer measures 2. A peptide carrying several extra protons appears at an m/z well below its mass, so a COA may list several m/z values for one molecule. Those values are usually converted back to a single neutral mass for comparison with the mass calculated from the sequence 3.

Is a mass spectrum the same as a purity result?

No. A mass spectrum shows which ions are present, but signal size depends on how readily each species ionises, and mobile-phase additives such as TFA can suppress ionisation 4. The EMA guideline treats identity and purity as separate specification tests, with purity usually measured by liquid chromatography 1. See HPLC purity explained for how that number is produced.

Can mass spectrometry tell leucine from isoleucine?

Not by mass alone. Leucine and isoleucine share the formula C6H13NO2 and have identical monoisotopic masses in PubChem 7. Medzihradszky and Chalkley note that the low-energy fragmentation used by most tandem instruments cannot tell the two residues apart, although high-energy CID can in principle 10. The EMA guideline lists NMR, which can identify individual amino acids, among other identity methods 1.

Why does one peptide give several peaks in an ESI spectrum?

Electrospray ionisation adds different numbers of protons to different copies of the same molecule. Fenn and colleagues described the result in 1989: a series of peaks from multiply charged ions, each differing from its neighbours by one charge 3. Software combines the series into one neutral mass. Sodium adducts, 21.98 Da heavier than the matching protonated ion, can add further peaks 9.

Is mass spectrometry enough on its own to confirm identity?

For medicines, European guidance says no. The EMA guideline recommends at least two orthogonal identification methods, chosen so that together they unambiguously confirm the sequence 1. Mass alone cannot separate leucine from isoleucine, or D- from L-amino acids 7, so MS/MS, amino acid analysis, NMR or chiral methods are added. Our guide to reading a peptide COA shows where identity results appear.

References

1. European Medicines Agency. Guideline on the Development and Manufacture of Synthetic Peptides (EMA/CHMP/CVMP/QWP/367182/2025), adopted 4 December 2025, effective 1 June 2026. https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-development-manufacture-synthetic-peptides_en.pdf. Accessed 28 September 2026. [regulatory guideline]
2. United States Pharmacopeia. General Chapter <736> Mass Spectrometry. USP-NF. doi:10.31003/USPNF_M99525_02_01. Accessed 28 September 2026. [pharmacopoeial chapter]
3. Fenn JB, Mann M, Meng CK, et al. Electrospray ionization for mass spectrometry of large biomolecules. Science. 1989;246(4926):64-71. doi:10.1126/science.2675315. PMID: 2675315. [review of analytical method]
4. Sikora K, Jaśkiewicz M, Neubauer D, et al. The Role of Counter-Ions in Peptides-An Overview. Pharmaceuticals (Basel). 2020;13(12):E442. doi:10.3390/ph13120442. PMID: 33287352. PMCID: PMC7761850. [narrative review]
5. Knochenmuss R. Ion formation mechanisms in UV-MALDI. Analyst. 2006;131(9):966-986. doi:10.1039/b605646f. PMID: 17047796. [narrative review]
6. 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. [analytical method study]
7. PubChem. Compound records for L-leucine (CID 6106), L-isoleucine (CID 6306), L-glutamine (CID 5961), L-lysine (CID 5962), L-alanine (CID 5950) and D-alanine (CID 71080): molecular formula, molecular weight and monoisotopic mass. https://pubchem.ncbi.nlm.nih.gov/compound/6106, https://pubchem.ncbi.nlm.nih.gov/compound/6306, https://pubchem.ncbi.nlm.nih.gov/compound/5961, https://pubchem.ncbi.nlm.nih.gov/compound/5962, https://pubchem.ncbi.nlm.nih.gov/compound/5950, https://pubchem.ncbi.nlm.nih.gov/compound/71080. Accessed 28 September 2026. [chemistry database]
8. Zubarev R, Mann M. On the proper use of mass accuracy in proteomics. Mol Cell Proteomics. 2007;6(3):377-381. doi:10.1074/mcp.m600380-mcp200. PMID: 17164402. [methods perspective]
9. Unimod. Records for Oxidation (accession 35), Deamidated (accession 7) and Cation:Na (accession 30). https://www.unimod.org/modifications_view.php?editid1=35, https://www.unimod.org/modifications_view.php?editid1=7, https://www.unimod.org/modifications_view.php?editid1=30. Accessed 28 September 2026. [mass spectrometry database]
10. Medzihradszky KF, Chalkley RJ. Lessons in de novo peptide sequencing by tandem mass spectrometry. Mass Spectrom Rev. 2015;34(1):43-63. doi:10.1002/mas.21406. PMID: 25667941. PMCID: PMC4367481. [tutorial review]
11. Roepstorff P, Fohlman J. Proposal for a common nomenclature for sequence ions in mass spectra of peptides. Biomed Mass Spectrom. 1984;11(11):601. doi:10.1002/bms.1200111109. PMID: 6525415. [letter]
12. Lian Z, Wang N, Tian Y, et al. Characterization of Synthetic Peptide Therapeutics Using Liquid Chromatography-Mass Spectrometry: Challenges, Solutions, Pitfalls, and Future Perspectives. J Am Soc Mass Spectrom. 2021;32(8):1852-1860. doi:10.1021/jasms.0c00479. PMID: 34110145. [narrative review]
13. 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. [narrative review]
14. Li M, Josephs RD, Daireaux A, et al. Identification and accurate quantification of structurally related peptide impurities in synthetic human C-peptide by liquid chromatography-high resolution mass spectrometry. Anal Bioanal Chem. 2018;410(20):5059-5070. doi:10.1007/s00216-018-1155-y. PMID: 29862433. [analytical study]
15. Currier JR, Galley LM, Wenschuh H, et al. Peptide impurities in commercial synthetic peptides and their implications for vaccine trial assessment. Clin Vaccine Immunol. 2008;15(2):267-276. doi:10.1128/cvi.00284-07. PMID: 18077621. PMCID: PMC2238048. [laboratory study, human T-cell assays]