Elucidation of chemical modifier reactivity towards peptides and proteins and the analysis of specific fragmentation by matrix-assisted laser desorption/ionization collision-induced dissociation tandem mass spectrometry

Rapid Commun Mass Spectrom. 2019 May;33 Suppl 1:40-49. doi: 10.1002/rcm.8223. Epub 2018 Aug 12.


Rationale: Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of covalent 5-lipoxygenase inhibitors is challenging due to unknown amino acid specificity and low posttranslational modification (PTM)-identification rates. The analysis of the amino-acid specificity and of the characteristic fragmentation of chemically modified peptides is considered to improve knowledge for the analysis of chemically modified peptides and proteins by MALDI-MS.

Methods: Various compounds were used to investigate the modification of synthetic peptides carrying reactive amino acid residues. Mass spectra were recorded using a MALDI-LTQ Orbitrap XL for high-resolution mass spectrometry and ion trap MALDI-MS2 . UV-Vis-based reduction and radical scavenging analysis was conducted. The on-plate digestion method described by Rühl et al was utilized for modification-site analysis at 5-lipoxygenase.

Results: The analysis of amino-acid-specific reactivity revealed the reactivity of quinones towards cysteine residues and the potential occurrence of a subsequent oxidative process was observed by an UV-Vis-based reduction assay. MALDI collision-induced dissociation tandem mass spectrometry (CID-MS2 ) indicated a prominent fragmentation mechanism of modified cysteine and histidine residues. Fragmentation included highly abundant neutral-loss signals which could be used to identify new modifications induced by chemical modifiers at the cysteine-159 residue of 5-lipoxygenase.

Conclusions: Specificity and fragmentation analysis provides crucial information for the analysis of chemically modified cysteines and histidines by MALDI-MS. Elucidation of binding sites by MALDI-MS has been significantly improved using an easy-to-run peptide assay and gives background information for the analysis in the case of chemically modified 5-lipoxygenase.

MeSH terms

  • Binding Sites
  • Cysteine / analysis
  • Cysteine / chemistry
  • Cysteine / metabolism
  • Histidine / analysis
  • Histidine / chemistry
  • Histidine / metabolism
  • Lipoxygenase
  • Lipoxygenase Inhibitors
  • Peptide Fragments / analysis
  • Peptide Fragments / chemistry
  • Peptides / analysis
  • Peptides / chemistry*
  • Peptides / metabolism
  • Protein Binding
  • Proteins / analysis
  • Proteins / chemistry*
  • Proteins / metabolism
  • Quinones / chemistry
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods*
  • Tandem Mass Spectrometry / methods*


  • Lipoxygenase Inhibitors
  • Peptide Fragments
  • Peptides
  • Proteins
  • Quinones
  • Histidine
  • Lipoxygenase
  • Cysteine