Mass spectrometric analysis data of Pyruvate Kinase from E. histolytica

To Identify the purified recombinant protein, we have performed Mass spectrometric analysis to prove it as Pyruvate Kinase from E. histolytica Sample Preparation for Mass spectrometric studies The protein spot was manually excised from the SDS-PAGE gel, and it was de-stained with 50 mM sodium thiosulphate and 15 mM potassium ferricyanide, and reduced with 5 mM TCEP and was finally alkylated with 50 mM iodoacetamide in the dark at room temperature for 30 min. Further, the gel particles were digested with (0.02μg/μl) trypsin in 50 mM ammonium bicarbonate and incubated at 37°C for 16–18 h (overnight). Digests were cleaned using a C18 silica cartridge to remove the salt and dried using a speed-vacuum centrifuge. The dried pellet was resuspended in buffer A (5% acetonitrile, 0.1% formic acid). Mass Spectrometric analysis of peptide mixtures The experiment was performed using EASY-nLC 1000 system (Thermo Fisher Scientific) coupled to Thermo Fisher-Q Exactive equipped with nano-electrospray ion source. Peptide mixture (1.0µg) was resolved using 25 cm PicoFrit column (360µm outer diameter, 75µm inner diameter, 10µm tip) filled with 1.9 µm of C18-resin (Dr Maeisch, Germany). The peptides were loaded with buffer A and eluted with a 0–40% gradient of buffer B (95% acetonitrile, 0.1% formic acid) at a flow rate of 300 nl/min for 100 min. MS data were acquired using a data-dependent top 10 method dynamically choosing the most abundant precursor ions from the survey scan. Data Processing The RAW files generated were analyzed with Proteome Discoverer (v2.2) against the UniProt Entamoeba histolytica reference proteome database. For Sequest search, the precursor and fragment mass tolerances were set at 10 ppm and 0.5 Da, respectively. The protease used to generate peptides, i.e. enzyme specificity was set for trypsin/P (cleavage at the C terminus of “K/R: unless followed by “P”) along with maximum missed cleavages value of two. Carbamidomethyl on cysteine as fixed modification and oxidation of methionine and N-terminal acetylation were considered as variable modifications for database search. Both peptide spectrum match and protein false discovery rate were set to 0.01 FDR.

为鉴定该纯化重组蛋白，我们开展了质谱分析，证实其为溶组织内阿米巴（Entamoeba histolytica）的丙酮酸激酶。

### 质谱分析样品制备流程
从十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）凝胶中手动切取目的蛋白条带，经50 mM硫代硫酸钠与15 mM铁氰化钾溶液脱色，再以5 mM三(2-羧乙基)膦（TCEP）进行还原反应，最后于室温暗处用50 mM碘乙酰胺进行烷基化处理30分钟。随后将凝胶颗粒置于50 mM碳酸氢铵缓冲液中，以0.02μg/μl的胰蛋白酶进行酶解，于37℃孵育16~18小时（过夜）。酶解产物经C18硅胶柱去除盐离子，再通过真空离心浓缩仪干燥。干燥后的肽段沉淀用缓冲液A（5%乙腈、0.1%甲酸）重悬。

### 肽混合物质谱分析
实验采用搭载纳米电喷雾离子源的赛默飞世尔Q Exactive质谱仪，联用EASY-nLC 1000超高效液相色谱系统完成。取1.0μg肽段混合物，使用25 cm长的PicoFrit色谱柱（外径360μm，内径75μm，尖端内径10μm，填充1.9 μm C18树脂，德国Maeisch博士提供）进行分离。以缓冲液A上样，采用0~40%的缓冲液B（95%乙腈、0.1%甲酸）梯度洗脱，流速为300 nl/min，洗脱时长100分钟。质谱数据采用数据依赖型Top10采集模式，从全扫描谱图中动态选取丰度最高的前体离子进行碎裂分析。

### 数据处理
采用Proteome Discoverer（v2.2）软件对生成的RAW文件进行分析，检索数据库为UniProt溶组织内阿米巴参考蛋白质组数据库。使用Sequest搜索引擎时，前体离子质量容忍度设为10 ppm，碎片离子质量容忍度设为0.5 Da。酶切特异性设置为胰蛋白酶/P（即于赖氨酸（K）和精氨酸（R）的C端切割，除非其后紧邻脯氨酸（P）），最大允许漏切位点数量为2。半胱氨酸的氨基甲酰甲基化设为固定修饰，甲硫氨酸氧化及N端乙酰化设为可变修饰。肽谱匹配（PSM）与蛋白质水平的错误发现率（FDR）均设置为0.01。