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.

为鉴定纯化的重组蛋白，本研究采用质谱分析法以证实其为来自溶组织阿米巴（E. histolytica）的丙酮酸激酶。

样品制备：质谱研究样品的制备过程中，蛋白质点被手动从SDS-PAGE凝胶中切取，经50 mM硫代硫酸钠和15 mM高铁氰化钾脱色，并使用5 mM三羧乙二醇进行还原处理。随后，在室温下于黑暗中用50 mM碘代乙酰胺进行烷基化反应，持续30分钟。进一步地，凝胶颗粒用（0.02μg/μl）胰蛋白酶在50 mM碳酸氢铵中进行消化，并在37°C下孵育16-18小时（过夜）。消化液通过C18硅胶柱进行纯化以去除盐分，并使用高速离心机进行干燥。干燥后的沉淀物用缓冲液A（5%乙腈，0.1%甲酸）重新悬浮。

肽混合物的质谱分析：实验使用Thermo Fisher Scientific的EASY-nLC 1000系统，与配备纳米电喷雾电离源的Thermo Fisher-Q Exactive质谱仪联用。肽混合物（1.0µg）使用25 cm PicoFrit柱（外径360µm，内径75µm，尖端10µm）进行分离，该柱填充有1.9 µm的C18树脂（Dr Maeisch，德国）。肽通过缓冲液A加载，并以300 nl/min的流速使用缓冲液B（95%乙腈，0.1%甲酸）的0-40%梯度洗脱，持续100分钟。MS数据采用数据依赖性前10方法动态选择调查扫描中最丰富的前体离子。

数据处理：生成的RAW文件使用Proteome Discoverer（v2.2）与UniProt Entamoeba histolytica参考蛋白质组数据库进行比对分析。对于Sequest搜索，前体和碎片质量容差分别设置为10 ppm和0.5 Da。用于生成肽的蛋白酶，即酶特异性设置为胰蛋白酶/P（在“K/R”的C端切割，除非其后跟有“P”），最大缺失切割值设为两个。对于数据库搜索，以半胱氨酸上的碳酰胺甲基化为固定修饰，以及蛋氨酸的氧化和N端乙酰化为可变修饰。肽谱匹配和蛋白质假发现率均设置为0.01 FDR。