Sequencing peptides by tandem mass spectrometry

An intact mass confirms a peptide's composition. To confirm its sequence, the order of the residues, the peptide is broken apart inside the mass spectrometer and the pieces are weighed. This is tandem mass spectrometry (MS/MS). This article explains how peptides fragment, how a fragment ladder is read, which fragmentation methods suit which questions, and what MS/MS cannot settle.

Principle

In the first stage, the instrument selects one ion, the precursor, usually a protonated form of the peptide such as [M+2H]²⁺. It then gives that ion enough energy to break, and the second stage measures the masses of the fragments. Because peptides tend to break along the backbone, between residues, the fragments form series whose masses step up one residue at a time.

Fragment ion types

Breaking the backbone splits the peptide in two. By the nomenclature of Roepstorff and Fohlman, later refined by Biemann, fragments that keep the N-terminus are called a, b or c ions, and fragments that keep the C-terminus x, y or z ions, depending on which backbone bond broke. The number gives how many residues the fragment contains.

Fragmentation methods and the ions they give

Method	How energy is added	Main ions	Good for
CID (collision-induced dissociation)	Collisions with an inert gas	b and y	Routine sequencing of small, protonated peptides
HCD (higher-energy collisional dissociation)	Collisions at higher energy, in a separate cell	b and y, more y; low-mass immonium ions	High-resolution fragment spectra; marker ions
ETD (electron-transfer dissociation)	An electron transferred from a reagent anion to a multiply charged precursor	c and z	Longer, highly charged peptides; keeping labile modifications attached

Reading a ladder

The difference between two neighboring ions in a series equals the mass of one residue, which identifies it: 57.021 Da for glycine, 71.037 for alanine, 113.084 for leucine or isoleucine, and so on. Reading the b series from low to high mass gives the sequence from the N-terminus; reading the y series gives it from the C-terminus. The two series run in opposite directions and confirm each other.

A worked example: for KPV, the b ions are at 129.102 (Lys) and 226.155 (Lys-Pro), and the y ions at 118.086 (Val) and 215.139 (Pro-Val). The step from 129.102 to 226.155 is 97.053 Da, a proline. Every peptide reference entry lists its expected b and y ions, and the peptide calculator gives them for any linear sequence.

Real spectra are not complete ladders. Some bonds break readily (the bond before proline is a well-known example), others rarely, and ions lose water or ammonia. A sequence is confirmed when enough consecutive steps are seen in at least one series, ideally both.

De novo sequencing and database matching

For a known synthetic peptide the question is confirmation: do the observed fragments match those predicted for the stated sequence? That is simple and robust. Reading an unknown sequence from scratch, de novo sequencing, is harder, because gaps and ambiguous steps leave several candidate sequences. Proteomics avoids the problem by matching spectra against sequence databases, an approach that suits natural proteins rather than modified synthetic peptides.

What MS/MS cannot settle

• Leucine and isoleucine have the same residue mass, so a ladder step cannot tell them apart without specialized fragmentation.
• Stereochemistry. A D-residue gives the same fragments as its L-form. Chiral analysis is needed.
• Rings. In a peptide closed by a disulfide or a lactam bridge, a single backbone cleavage inside the ring does not release a fragment. Reduction before analysis, or more complex interpretation, is needed.
• Quantity. Fragment spectra confirm identity; they do not measure purity.

Frequently asked questions

Why is the doubly charged ion usually chosen as the precursor?

In electrospray, many peptides form [M+2H]²⁺ abundantly, and doubly charged precursors fragment into informative b and y ions spread across the spectrum.

What is an immonium ion?

A small ion formed from a single residue, its mass less carbon monoxide plus a proton. Immonium ions in the low-mass region indicate which amino acids are present, though not their order.

Does a matching fragment spectrum prove the material is pure?

No. It confirms the sequence of the ion that was selected. Purity is measured by chromatography.

Sources & Further Reading

1. Roepstorff P, Fohlman J. Letter to the editors: proposal for a common nomenclature for sequence ions in mass spectra of peptides. Biomedical Mass Spectrometry 1984;11(11):601. doi:10.1002/bms.1200111109
2. Biemann K. Contributions of mass spectrometry to peptide and protein structure. Biomedical and Environmental Mass Spectrometry 1988;16(1–12):99–111. doi:10.1002/bms.1200160119
3. Steen H, Mann M. The ABC's (and XYZ's) of peptide sequencing. Nature Reviews Molecular Cell Biology 2004;5(9):699–711. doi:10.1038/nrm1468
4. Paizs B, Suhai S. Fragmentation pathways of protonated peptides. Mass Spectrometry Reviews 2005;24(4):508–548. doi:10.1002/mas.20024
5. Syka JE, Coon JJ, Schroeder MJ, Shabanowitz J, Hunt DF. Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. Proceedings of the National Academy of Sciences of the United States of America 2004;101(26):9528–9533. doi:10.1073/pnas.0402700101

Cosmic Peptides. (2026, October 25). Sequencing peptides by tandem mass spectrometry. Cosmic Peptides Research Library. Retrieved October 26, 2026, from https://cosmicpeptides.com/research/methods/tandem-ms-sequencing

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  author       = {{Cosmic Peptides}},
  title        = {Sequencing peptides by tandem mass spectrometry},
  howpublished = {Cosmic Peptides Research Library},
  year         = {2026},
  month        = oct,
  url          = {https://cosmicpeptides.com/research/methods/tandem-ms-sequencing},
  urldate      = {2026-10-26},
  note         = {Last reviewed 2026-10-25}
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