Winning The Last Battle Against Edman Degradation: Reliable Leucine/Iso-leucine Differentiation In Peptide Sequencing Using an Orbitrap Fusion Mass Spectrometer

Importance of Differentiating Leucine and Isoleucine in Peptide Sequencing

Accurate discrimination of isomeric amino acids leucine and isoleucine is critical for reliable de novo peptide sequencing by mass spectrometry. These isomers share identical mass but produce distinct side chain fragmentations. Conventional MS2 approaches often fail to generate clear w ions and suffer from radical site migration along the peptide backbone. Implementing a targeted MS3 workflow on a high-resolution tribrid platform addresses these limitations and improves confidence in peptide identification.

Aims and Study Overview

The study aimed to develop a straightforward, robust method for unambiguous Leu/Ile differentiation within natural peptides up to 37 residues in length. Six peptides isolated from Rana ridibunda skin secretions, varying in basic residue content and number of isomeric sites, served as test substrates. The work focused on combining electron transfer dissociation (ETD) with higher energy collisional dissociation (HCD) in a sequential MS3 acquisition to generate selective w ion spectra.

Methodology

Peptides were directly infused and subjected to MS2 by ETD to produce odd-electron z ions retaining the N-terminal side chain. Selected z ions then underwent HCD fragmentation in MS3. Collision energies (NCE) were scanned from 10 to 40 to optimize side chain losses that distinguish isopropyl and ethyl radicals. Manual precursor selection ensured targeting of z ions carrying individual Leu or Ile residues. Key performance parameters included spectral selectivity and minimal interference from backbone radical migration.

Použitá instrumentace

• Orbitrap Fusion Tribrid mass spectrometer
• EASY-Max NG ion source in infusion mode
• Tune 1.0 software for MSn control

Main Results and Discussion

The ETD/HCD MS3 method delivered highly selective spectra dominated by precursor z ions and a small set of w product ions. The diagnostic mass differences of 29.039 and 43.054 Da from ethyl and isopropyl side chains allowed clear distinction of Ile versus Leu. Radical site migration was significantly reduced compared to ion trap techniques. Examples included single isomer sites in ranatuerin and brevinin peptides, as well as contiguous Ile-Leu-Leu motifs. In all cases the dominant w ion unambiguously indicated the correct isomer, even when neighboring residues could potentially undergo radical transfer at higher NCE.

Benefits and Practical Applications

• Enables direct de novo sequencing of non-tryptic and complex natural peptides
• Produces simplified MS3 spectra with one to four product ions
• Reduces ambiguity from radical migration along the peptide chain
• Applicable to high-throughput proteomic workflows requiring isomer resolution

Future Trends and Opportunities

Integration of this ETD/HCD MS3 strategy into automated acquisition methods and data analysis pipelines will further streamline de novo sequencing. Advances in instrument control and software algorithms could allow dynamic selection of optimal NCE values and real-time identification of isomeric residues. Extending the approach to larger proteins, modified peptides and cross-linked species represents a promising direction for next-generation proteomics.

Conclusion

The described ETD/HCD MS3 workflow on an Orbitrap Fusion platform offers a reliable, efficient solution for leucine and isoleucine differentiation in peptide sequencing. By exploiting characteristic side chain losses and minimizing radical migration artifacts, the method enhances confidence in de novo peptide characterization without recourse to orthogonal chemical derivatization.

Reference

1. Kjeldsen F et al Anal Chem 2003
2. Hongling H et al J Proteome Res 2007
3. Gupta K et al J Proteome Res 2012
4. Leymarie N et al J Am Chem Soc 2003