Combined electron transfer dissociation-collision-induced dissociation fragmentation in the mass spectrometric distinction of leucine, isoleucine, and hydroxyproline residues in Peptide natural products

J Proteome Res. 2012 Feb 3;11(2):515-22. doi: 10.1021/pr200091v. Epub 2011 Dec 28.


Distinctions between isobaric residues have been a major challenge in mass spectrometric peptide sequencing. Here, we propose a methodology for distinction among isobaric leucine, isoleucine, and hydroxyproline, a commonly found post-translationally modified amino acid with a nominal mass of 113 Da, through a combined electron transfer dissociation-collision-induced dissociation approach. While the absence of c and z(•) ions, corresponding to the Yyy-Xxx (Xxx = Leu, Ile, or Hyp) segment, is indicative of the presence of hydroxyproline, loss of isopropyl (Δm = 43 Da) or ethyl radicals (Δm = 29 Da), through collisional activation of z radical ions, are characteristic of leucine or isoleucine, respectively. Radical migration processes permit distinctions even in cases where the specific z(•) ions, corresponding to the Yyy-Leu or -Ile segments, are absent or of low intensity. This tandem mass spectrometric (MS(n)) method has been successfully implemented in a liquid chromatography-MS(n) platform to determine the identity of 23 different isobaric residues from a mixture of five different peptides. The approach is convenient for distinction of isobaric residues from any crude peptide mixture, typically encountered in natural peptide libraries or proteomic analysis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Biological Products / chemistry*
  • Hydroxyproline / chemistry*
  • Isoleucine / chemistry*
  • Leucine / chemistry*
  • Mass Spectrometry / methods*
  • Melitten / chemistry
  • Molecular Sequence Data
  • Peptaibols / chemistry
  • Peptides / chemistry*
  • Sequence Analysis, Protein / methods*
  • Wasp Venoms / chemistry


  • Biological Products
  • Peptaibols
  • Peptides
  • Wasp Venoms
  • Isoleucine
  • antiamoebin
  • Melitten
  • zervamicin IIB
  • Leucine
  • Hydroxyproline