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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

 
: Rühl, M.; Kühn, B.; Roos, J.; Maier, T.J.; Steinhilber, D.; Karas, M.

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Rapid communications in mass spectrometry : RCM 33 (2019), No.S1, pp.40-49
ISSN: 0951-4198
ISSN: 1097-0231
European Mass Spectrometry Conference (EMSC) <1, 2018, Saarbrücken>
English
Journal Article, Conference Paper
Fraunhofer IME ()

Abstract
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.

: http://publica.fraunhofer.de/documents/N-541072.html