Determining the exact amino acid sequence of a peptide is fundamental to its characterization. Multiple techniques exist, ranging from classical chemical methods to cutting-edge mass spectrometric approaches.
Edman Degradation
The Classical Method
Developed by Pehr Edman in 1949, this technique sequentially removes and identifies amino acids from the N-terminus.
Process
- Coupling — phenylisothiocyanate (PITC) reacts with the N-terminal amino acid
- Cleavage — mild acid cleaves the modified N-terminal residue as a thiazolinone
- Conversion — the thiazolinone is converted to a stable PTH-amino acid
- Identification — HPLC identifies the PTH-amino acid
- Repeat — the cycle continues with the next amino acid
Limitations
- Requires a free N-terminus (blocked termini cannot be sequenced)
- Practical limit of ~50-60 residues due to cumulative inefficiency
- Cannot distinguish leucine from isoleucine
- Relatively slow (one residue per 30-60 minutes)
Mass Spectrometry-Based Sequencing
De Novo Sequencing
Using MS/MS fragmentation data to determine sequence without reference databases:
- Peptide ions are selected and fragmented
- b-ion and y-ion series are identified
- Mass differences between consecutive ions reveal amino acid identity
- The complete sequence is assembled
Database Searching
For known peptides, MS/MS spectra can be matched against theoretical fragmentation patterns from sequence databases.
Amino Acid Analysis (AAA)
Composition (Not Sequence)
While not providing sequence information, AAA determines the amino acid composition:
- Hydrolysis — peptide bonds are broken by acid (6N HCl, 110°C, 24 hours)
- Derivatization — free amino acids are derivatized for detection
- Separation — ion-exchange or RP-HPLC separates the derivatized amino acids
- Quantification — peak areas determine the amount of each amino acid
Value
- Confirms expected amino acid ratios
- Detects unexpected amino acids
- Provides accurate concentration measurements
Modern Integrated Approaches
Today's peptide sequencing typically combines multiple techniques:
- MALDI-TOF for molecular weight confirmation
- LC-MS/MS for sequence verification
- Edman degradation for resolving ambiguities (especially Leu/Ile)
- AAA for composition verification
Ensuring Sequence Accuracy at Evolve Aminos
Our quality control employs both mass spectrometry-based sequencing and complementary techniques to guarantee that every peptide's sequence is precisely as specified.