Institute of Technical Biochemistry, Lodz University of Technology, 4/10 Stefanowskiego Street, 90-924, Lodz, Poland.
Amino Acids. 2019 Sep;51(9):1365-1375. doi: 10.1007/s00726-019-02773-8. Epub 2019 Aug 30.
The regulatory role of protein cysteine phosphorylation is an under-researched area. The difficulty of accessing reference S-phosphorylated peptides (pCys-peptides) hampers progress in MS-driven cysteine phosphoproteomics, which requires targeted analytical procedures. This work describes an uncomplicated process for the conversion of disulfide-bridged protein into a complex model mixture of combinatorially modified peptides. Hen egg-white lysozyme was reduced with tris(2-carboxyethyl)phosphine (TCEP) followed by alkylation of cysteine with (3-acrylamidopropyl)trimethyl-ammonium chloride (APTA) and subsequent beta-elimination in aqueous Ba(OH) to yield modified polypeptides containing multiple dehydroalanine (Dha) residues. The conjugate addition of thiophosphoric acid to Dha residues followed by trypsinolysis led to numerous D/L phosphocysteine-containing peptides, which were identified by higher-energy collisional-dissociation tandem mass spectrometry (HCD-MS/MS). Our results show that some pCys-peptides produce prominent neutral losses of 80 Da, 98 Da and a weak 116 Da loss. These are similar to the neutral-loss triplets generated by phosphohistidine peptides.
蛋白质半胱氨酸磷酸化的调控作用是一个研究不足的领域。由于难以获得参考 S-磷酸化肽（pCys-肽），阻碍了基于 MS 的半胱氨酸磷酸化蛋白质组学的发展，这需要有针对性的分析程序。本工作描述了一种将二硫键桥连蛋白质转化为组合修饰肽复杂模型混合物的简单方法。用三(2-羧乙基)膦（TCEP）还原鸡卵清白溶菌酶，然后用（3-丙烯酰胺丙基）三甲基氯化铵（APTA）对半胱氨酸进行烷基化，随后在 Ba(OH)2 水溶液中进行β消除，得到含有多个脱氢丙氨酸（Dha）残基的修饰多肽。Dha 残基与硫代磷酸的共轭加成，随后用胰蛋白酶酶解，产生了许多含有 D/L 磷酸半胱氨酸的肽，这些肽通过高能碰撞解离串联质谱（HCD-MS/MS）进行了鉴定。我们的结果表明，一些 pCys-肽会产生明显的 80 Da、98 Da 和较弱的 116 Da 中性丢失。这些与磷酸组氨酸肽产生的中性丢失三胞胎相似。
