Tandem mass spectrometry (MS/MS) deliberately fragments peptide ions to generate structural information. Understanding fragmentation patterns is essential for interpreting spectra and confirming peptide sequences.
Peptide Bond Fragmentation
Nomenclature
Peptide fragmentation follows the Roepstorff-Fohlman-Biemann nomenclature:
N-terminal fragments (charge retained on N-terminus):
- a ions ā cleavage at CĪ±-CO bond
- b ions ā cleavage at CO-NH bond (most common)
- c ions ā cleavage at NH-CĪ± bond
C-terminal fragments (charge retained on C-terminus):
- x ions ā cleavage at CĪ±-CO bond
- y ions ā cleavage at CO-NH bond (most common)
- z ions ā cleavage at NH-CĪ± bond
Dominant Fragment Types
In collision-induced dissociation (CID), the most common fragmentation mode:
- b and y ions dominate the spectrum
- Their mass differences correspond to individual amino acid residues
- A complete series of b or y ions allows full sequence determination
Reading Fragmentation Spectra
Sequence Determination
- Identify the b-ion or y-ion series
- Calculate mass differences between consecutive ions
- Match mass differences to amino acid residue weights
- Assemble the sequence from the series
Common Mass Differences
| Amino Acid | Residue Mass (Da) |
|---|---|
| Glycine (G) | 57.02 |
| Alanine (A) | 71.04 |
| Valine (V) | 99.07 |
| Leucine/Isoleucine (L/I) | 113.08 |
| Phenylalanine (F) | 147.07 |
| Tryptophan (W) | 186.08 |
Note: Leucine and isoleucine are isobaric and cannot be distinguished by standard CID.
Special Fragmentation Features
Proline Effect
Proline residues create preferential cleavage at the N-terminal side, producing enhanced y-ions.
Neutral Losses
- Loss of water (ā18 Da) from Ser, Thr, Asp, Glu
- Loss of ammonia (ā17 Da) from Asn, Gln, Lys, Arg
- These provide additional structural information
Immonium Ions
Low-mass ions diagnostic for specific amino acids (e.g., m/z 86 for Leu/Ile, m/z 120 for Phe).
Advanced Fragmentation Methods
Electron Transfer Dissociation (ETD)
- Produces c and z ions
- Better for larger peptides and post-translational modifications
- Complementary to CID
Higher-Energy Collisional Dissociation (HCD)
- Similar to CID but with more complete fragmentation
- Standard on Orbitrap instruments
Quality Control Application
At Evolve Aminos, MS/MS fragmentation analysis is used to unambiguously confirm peptide sequences, ensuring that every product matches its specified amino acid sequence exactly.