Elucidation of chemical modifier reactivity towards peptides and proteins and the analysis of specific fragmentation by MALDI-CID-MS .

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 Ruhl 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.