MS/MS Fragmentation of Peptides

Tandem mass spectrometry selects a precursor peptide ion and fragments it to generate sequence-informative product ions.

Backbone cleavage

Common collision-based methods produce fragment series from cleavage along the peptide backbone. These fragments can be matched to theoretical sequence ions.

Fragment coverage

Coverage depends on:

• precursor charge
• collision energy
• sequence
• modifications
• instrument type
• fragment mass range
• signal intensity

Modification localization

A mass shift retained on specific fragments can help localize oxidation, deamidation, conjugation, or other modifications.

Limitations

Incomplete coverage, neutral losses, internal fragments, isobaric residues, and labile modifications can complicate interpretation.

Frequently asked questions

Can MS/MS distinguish leucine from isoleucine?

Routine fragmentation generally cannot distinguish them confidently.

Does full sequence coverage guarantee stereochemistry?

No. D- and L-residues have the same mass.

Why use multiple fragmentation methods?

Different methods provide complementary sequence information.

Can software identify every fragment correctly?

No. Expert review and validation remain important.

Key takeaways

MS/MS provides sequence-level evidence, but interpretation depends on coverage, fragmentation behavior, and known analytical limitations.

References

1. Aebersold R, Mann M. Mass-spectrometric exploration of proteome structure and function. Nature. 2016.
2. Gross JH. Mass Spectrometry: A Textbook. Springer.

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TSMS Labs educational disclaimer: For laboratory research and educational purposes only. Not for human consumption. This content is not medical, clinical, or regulatory advice.