An EThcD-Based Method for Discrimination of Leucine and Isoleucine Residues in Tryptic Peptides

J Am Soc Mass Spectrom. 2017 Aug;28(8):1600-1611. doi: 10.1007/s13361-017-1674-3. Epub 2017 Apr 26.


An EThcD-based approach for the reliable discrimination of isomeric leucine and isoleucine residues in peptide de novo sequencing procedure has been proposed. A multistage fragmentation of peptide ions was performed with Orbitrap Elite mass spectrometer in electrospray ionization mode. At the first stage, z-ions were produced by ETD or ETcaD fragmentation of doubly or triply charged peptide precursor ions. These primary ions were further fragmented by HCD with broad-band ion isolation, and the resulting w-ions showed different mass for leucine and isoleucine residues. The procedure did not require manual isolation of specific z-ions prior to HCD stage. Forty-three tryptic peptides (3 to 27 residues) obtained by trypsinolysis of human serum albumin (HSA) and gp188 protein were analyzed. To demonstrate a proper solution for radical site migration problem, three non-tryptic peptides were also analyzed. A total of 93 leucine and isoleucine residues were considered and 83 of them were correctly identified. The developed approach can be a reasonable substitution for additional Edman degradation procedure, which is still used in peptide sequencing for leucine and isoleucine discrimination. Graphical Abstract ᅟ.

Keywords: EThcD; Leucine/isoleucine differentiation; Orbitrap; Peptide sequencing; Tryptic peptides.

MeSH terms

  • Amino Acid Sequence
  • Amphibian Proteins / chemistry
  • Animals
  • Antimicrobial Cationic Peptides / chemistry
  • Humans
  • Isoleucine / analysis*
  • Leucine / analysis*
  • Mass Spectrometry / methods*
  • Peptide Fragments / chemistry*
  • Rana ridibunda
  • Sequence Analysis, Protein / methods
  • Serum Albumin, Human / chemistry*


  • Amphibian Proteins
  • Antimicrobial Cationic Peptides
  • Peptide Fragments
  • Isoleucine
  • brevinin-2, Rana
  • Leucine
  • Serum Albumin, Human