Global Quantitative SILAC Phosphoproteomics Reveals Differential Phosphorylation Is Widespread between the Procyclic and Bloodstream Form Lifecycle Stages of <i>Trypanosoma brucei</i>

We report a global quantitative phosphoproteomic study of bloodstream and procyclic form Trypanosoma brucei using SILAC labeling of each lifecycle stage. Phosphopeptide enrichment by SCX and TiO2 led to the identification of a total of 10096 phosphorylation sites on 2551 protein groups and quantified the ratios of 8275 phosphorylation sites between the two lifecycle stages. More than 9300 of these sites (92%) have not previously been reported. Model-based gene enrichment analysis identified over representation of Gene Ontology terms relating to the flagella, protein kinase activity, and the regulation of gene expression. The quantitative data reveal that differential protein phosphorylation is widespread between bloodstream and procyclic form trypanosomes, with significant intraprotein differential phosphorylation. Despite a lack of dedicated tyrosine kinases, 234 phosphotyrosine residues were identified, and these were 3–4 fold over-represented among site changing >10-fold between the two lifecycle stages. A significant proportion of the T. brucei kinome was phosphorylated, with evidence that MAPK pathways are functional in both lifecycle stages. Regulation of gene expression in T. brucei is exclusively post-transcriptional, and the extensive phosphorylation of RNA binding proteins observed may be relevant to the control of mRNA stability in this organism.本研究针对血流型与前循环型布氏锥虫（Trypanosoma brucei）开展了全局定量磷酸化蛋白质组学研究，对两个生活史阶段分别采用细胞培养稳定同位素氨基酸标记（Stable Isotope Labeling by Amino acids in Cell culture，SILAC）进行标记。通过强阳离子交换色谱（Strong Cation Exchange Chromatography，SCX）与二氧化钛（TiO₂）实现磷酸肽富集后，本研究共鉴定得到2551个蛋白质群对应的10096个磷酸化位点，并定量了两个生活史阶段间8275个磷酸化位点的表达比值。其中超过9300个位点（占比92%）此前未见报道。基于模型的基因富集分析显示，与鞭毛、蛋白激酶活性以及基因表达调控相关的基因本体论（Gene Ontology，GO）术语出现显著富集。定量数据表明，血流型与前循环型锥虫之间普遍存在蛋白质磷酸化差异，且存在显著的蛋白质内磷酸化差异。尽管布氏锥虫缺乏专一性酪氨酸激酶，本研究仍鉴定出234个磷酸酪氨酸残基，且在两个生活史阶段间表达变化超过10倍的位点中，这类残基的富集程度达到3~4倍。布氏锥虫激酶组（kinome）中有相当比例的成员发生了磷酸化，且有证据显示丝裂原活化蛋白激酶（Mitogen-Activated Protein Kinase，MAPK）通路在两个生活史阶段均具有功能。布氏锥虫的基因表达调控完全为转录后调控，本研究中观察到的RNA结合蛋白（RNA binding protein）广泛磷酸化现象，可能与该生物体内mRNA稳定性的调控密切相关。