Opposite Electron-Transfer Dissociation and Higher-Energy Collisional Dissociation Fragmentation Characteristics of Proteolytic K/R(X)n and (X)nK/R Peptides Provide Benefits for Peptide Sequencing in Proteomics and Phosphoproteomics

J Proteome Res. 2017 Feb 3;16(2):852-861. doi: 10.1021/acs.jproteome.6b00825. Epub 2016 Dec 2.


A key step in shotgun proteomics is the digestion of proteins into peptides amenable for mass spectrometry. Tryptic peptides can be readily sequenced and identified by collision-induced dissociation (CID) or higher-energy collisional dissociation (HCD) because the fragmentation rules are well-understood. Here, we investigate LysargiNase, a perfect trypsin mirror protease, because it cleaves equally specific at arginine and lysine residues, albeit at the N-terminal end. LysargiNase peptides are therefore practically tryptic-like in length and sequence except that following ESI, the two protons are now both positioned at the N-terminus. Here, we compare side-by-side the chromatographic separation properties, gas-phase fragmentation characteristics, and (phospho)proteome sequence coverage of tryptic (i.e., (X)nK/R) and LysargiNase (i.e., K/R(X)n) peptides using primarily electron-transfer dissociation (ETD) and, for comparison, HCD. We find that tryptic and LysargiNase peptides fragment nearly as mirror images. For LysargiNase predominantly N-terminal peptide ions (c-ions (ETD) and b-ions (HCD)) are formed, whereas for trypsin, C-terminal fragment ions dominate (z-ions (ETD) and y-ions (HCD)) in a homologous mixture of complementary ions. Especially during ETD, LysargiNase peptides fragment into low-complexity but information-rich sequence ladders. Trypsin and LysargiNase chart distinct parts of the proteome, and therefore, the combined use of these enzymes will benefit a more in-depth and reliable analysis of (phospho)proteomes.

Keywords: LysargiNase; electron-transfer dissociation; higher-energy collisional dissociation; peptide fragmentation; phosphoproteomics; proteomics; trypsin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Electrons*
  • Kinetics
  • Metalloproteases / chemistry*
  • Peptide Fragments / analysis*
  • Phosphoproteins / chemistry*
  • Protein Binding
  • Protein Conformation, alpha-Helical
  • Protein Conformation, beta-Strand
  • Protein Domains
  • Proteolysis
  • Proteomics / methods
  • Protons*
  • Sequence Analysis, Protein
  • Thermodynamics
  • Trypsin / chemistry*


  • Peptide Fragments
  • Phosphoproteins
  • Protons
  • Metalloproteases
  • ulilysin, Methanosarcina acetivorans
  • Trypsin