Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Straße 9, 60438, Frankfurt/Main, Germany.
Clinic of Anesthesiology, Intensive Care Medicine and Pain Therapy, Goethe University Hospital, Theodor-Stern-Kai 7, 60590, Frankfurt/Main, Germany.
Rapid Commun Mass Spectrom. 2019 May;33 Suppl 1:40-49. doi: 10.1002/rcm.8223. Epub 2018 Aug 12.
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.
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-MS . 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.
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-MS ) 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.
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.
由于未知的氨基酸特异性和低翻译后修饰 (PTM) 鉴定率，基质辅助激光解吸/电离质谱 (MALDI-MS) 分析共价 5-脂氧合酶抑制剂具有挑战性。化学修饰肽的氨基酸特异性和特征性片段分析被认为可以提高通过 MALDI-MS 分析化学修饰肽和蛋白质的知识。
使用各种化合物来研究带有反应性氨基酸残基的合成肽的修饰。使用 MALDI-LTQ Orbitrap XL 进行高分辨率质谱和离子阱 MALDI-MS 记录质谱。进行基于 UV-Vis 的还原和自由基清除分析。使用 Rühl 等人描述的板上消化方法在 5-脂氧合酶上进行修饰位点分析。
氨基酸特异性反应性分析揭示了醌类化合物对半胱氨酸残基的反应性，并且通过基于 UV-Vis 的还原测定观察到随后发生的氧化过程的可能性。MALDI 碰撞诱导解离串联质谱 (CID-MS) 表明修饰的半胱氨酸和组氨酸残基的主要碎裂机制。片段化包括高度丰富的中性丢失信号，可用于鉴定化学修饰剂在 5-脂氧合酶的半胱氨酸 159 残基上诱导的新修饰。
特异性和片段分析为 MALDI-MS 分析化学修饰的半胱氨酸和组氨酸提供了关键信息。使用易于运行的肽测定法显著提高了 MALDI-MS 对结合位点的阐明，并为化学修饰 5-脂氧合酶的分析提供了背景信息。
