Comparative phosphoproteome of Trypanosoma brucei bloodstream & procyclic form

Comparative phosphoproteomic analysis of SILAC labelled cultured bloodstream and procyclic form Trypansoma brucei. Phosphopeptide enrichement via SCX and TiO2, acquired on Oribtrap Velos using MSA. Triplicate biological replicate for each lifecycle stage (as unmixed, and two SILAC label swap expreiments), eight fraction injected in technical triplicate. For the SILAC labelled samples, the corresponding changes in the proteome were measured using unenriched peptides sperated by SCX. Data was processed using MaxQuant version 1.3.0.5 which incorporates the Andromeda search engine. Proteins were identified by searching a protein sequence database containingT. brucei brucei 927 annotated proteins (Version 4.0, downloaded from TriTrypDB http://www.tritrypdb.org/ supplemented with the VSG221 sequence and frequently observed contaminants (porcine trypsin, bovine serum albumins and mammalian keratins) that contains a total of 10,081 protein sequences. Search parameters specified an MS tolerance of 6 ppm, an MS/MS tolerance at 0.5 Da and full trypsin specificity, allowing for up to two missed cleavages. Carbamidomethylation of cysteine was set as a fixed modification and oxidation of methionines, N-terminal protein acetylation and N-pyroglutamate were allowed as variable modifications. Phosphoproteomic analysis included phosophorylation of serine, threonine and tyrosine as additional variable modifications. Peptides were required to be at least 7 amino acids in length and a MaxQuant score >5, with false discovery rates (FDRs) of 0.01 calculated at the levels of peptides, proteins and modification sites based on the number of hits against the reversed sequence database.

针对经细胞培养稳定同位素氨基酸标记（Stable Isotope Labeling by Amino Acids in Cell Culture，SILAC）培养的布氏锥虫（Trypanosoma brucei）血流型与前循环型菌株的比较磷酸化蛋白质组学分析。通过强阳离子交换色谱（Strong Cation Exchange Chromatography，SCX）与二氧化钛（Titanium Dioxide，TiO₂）完成磷酸肽富集，采用轨道阱Velos质谱仪（Orbitrap Velos）结合多级活化（Multistage Activation，MSA）模式进行数据采集。每个生命周期阶段设置三次生物学重复（包含未混合样本与两次SILAC标记互换实验），每份样本分为8个组分，每个组分进行三次技术重复进样。对于经SILAC标记的样本，同步采用未富集肽段经SCX分离的方式检测其蛋白质组的相应变化。数据处理使用整合了Andromeda搜索引擎的MaxQuant 1.3.0.5版本软件。蛋白质鉴定基于布氏锥虫布氏亚种927株的注释蛋白质序列数据库（版本4.0，下载自TriTrypDB数据库http://www.tritrypdb.org，补充VSG221序列及常见污染物序列：猪胰蛋白酶、牛血清白蛋白与哺乳动物角蛋白，总计包含10081条蛋白质序列）。检索参数设置如下：母离子质量偏差容忍度为6 ppm，子离子质量偏差容忍度为0.5 Da，采用严格胰蛋白酶酶切特异性，允许最多两次漏切位点；半胱氨酸氨基甲酰化为固定修饰，甲硫氨酸氧化、蛋白质N端乙酰化与N端焦谷氨酸化为可变修饰；磷酸化蛋白质组分析额外将丝氨酸、苏氨酸与酪氨酸的磷酸化设为可变修饰。肽段长度需至少为7个氨基酸残基，MaxQuant得分需大于5；基于反向序列数据库的匹配结果，在肽段、蛋白质与修饰位点水平分别计算得到0.01的假发现率（False Discovery Rate，FDR）。