Department of Analytical Biochemistry and Interfaculty Mass Spectrometry Center, University of Groningen , A. Deusinglaan 1, 9713 AV, Groningen, The Netherlands.
Department of Pediatrics, Center for Liver, Digestive and Metabolic Diseases, University Medical Center Groningen, University of Groningen , Hanzeplein 1, 9713 GZ, Groningen, The Netherlands.
Anal Chem. 2017 Jul 5;89(13):7123-7129. doi: 10.1021/acs.analchem.7b01039. Epub 2017 Jun 19.
Specific digestion of proteins is an essential step for mass spectrometry-based proteomics, and the chemical labeling of the resulting peptides is often used for peptide enrichment or the introduction of desirable tags. Electrochemical oxidation yielding specific cleavage C-terminal to tyrosine (Tyr) and tryptophan (Trp) residues provides a potential alternative to enzymatic digestion and a possibility for further chemical labeling by introducing reactive spirolactone moieties. However, spirolactone-containing peptides suffer from low stability due to hydrolysis and intramolecular side reactions. We found that Cu(II) ions stabilize the spirolactone and prevent intramolecular side reactions during chemical labeling, allowing efficient chemical tagging with a reduced excess of labeling reagent without intramolecular side reactions. On the basis of this reaction, we developed an analytical procedure combining electrochemical digestion, Cu(II)-mediated spirolactone biotinylation, and enrichment by avidin affinity chromatography with mass spectrometry. The method was optimized with the tripeptide LWL and subsequently applied to chicken egg white lysozyme, in which one biotinylated electrochemistry (EC)-cleaved peptide was identified after affinity enrichment. This proof-of-principle shows that specific enrichment of electrochemically cleaved spirolactone-containing peptides can be used for protein identification and notably that inclusion of Cu(II) ions is essential for stabilizing spirolactones for subsequent biotinylation.
蛋白质的特异性消化是基于质谱的蛋白质组学的一个重要步骤，而生成的肽的化学标记通常用于肽的富集或引入所需的标记物。电化学氧化产生的酪氨酸（Tyr）和色氨酸（Trp）残基的特异性裂解为酶解提供了一种潜在的替代方法，并且通过引入反应性螺内酯部分为进一步的化学标记提供了可能性。然而，由于水解和分子内副反应，含螺内酯的肽稳定性差。我们发现 Cu(II) 离子稳定了螺内酯并防止了化学标记过程中的分子内副反应，从而可以在没有分子内副反应的情况下，用减少的过量标记试剂进行有效的化学标记。基于该反应，我们开发了一种分析程序，该程序将电化学消化、Cu(II)介导的螺内酯生物素化以及与质谱结合的亲和层析的亲和浓缩相结合。该方法用三肽 LWL 进行了优化，随后应用于鸡卵清白溶菌酶，在亲和富集后鉴定出一个生物素化的电化学（EC）切割肽。该原理验证表明，电化学切割的含螺内酯肽的特异性富集可用于蛋白质鉴定，特别是 Cu(II) 离子的包含对于螺内酯的后续生物素化的稳定是必不可少的。
