液质联用鉴定重组人1型单纯疱疹病毒载体的蛋白种类

目的： 采用液质联用技术鉴定重组人1型单纯疱疹病毒载体中的蛋白种类。方法： 用Triton X-100裂解病毒，用丙酮提取病毒蛋白；将病毒蛋白还原烷基化后用胰蛋白酶酶切，酶切肽段进行液质联用分析。色谱柱为C₁₈（2.1 mm×150 mm，1.7 μm），以含0.1%甲酸的水溶液（A）-含0.1%甲酸的乙腈溶液（B）为流动相，梯度洗脱，流速0.3 mL·min^-1。质谱仪采用正离子、高灵敏度、IDA模式采集数据，喷雾电压5.5 kV，离子源温度550℃，扫描范围m/z 200~2 000；将采集的质谱数据导入到ProteinPilot软件中进行搜库检索，肽段与蛋白的可能性大于95%时可认为鉴定，人工对匹配的串联图谱进行确证，肽段及蛋白可接受的假阳性率（FDR）不高于1%。结果： 在人单纯疱疹病毒蛋白数据库中检索，鉴定到29种人单纯疱疹病毒蛋白，大多为衣壳蛋白及包膜蛋白；在绿猴（Chlorocebus）属蛋白数据库中检索，鉴定到2种宿主细胞蛋白；在全病毒蛋白数据库中检索，鉴定到1种猫肉瘤病毒蛋白。结论： 液质联用技术具有高灵敏度、高分辨率的特点，可用于基因治疗产品的质量研究。

Objective: To identify the proteins in the sample of recombinant human herpes simplex virus type 1 by liquid chromatography-mass spectrometry(LC-MS). Methods: The virus was spited with Triton X-100 and the viral protein was extracted with acetone. The purified proteins were reduced and alkylated,and then digested by trypsin and analyzed by LC-MS. The chromatographic separation was carried out on a C₁₈ column(2.1 mm×150 mm,1.7 μm) with the mobile phase consisting of water containing 0.1% formic acid(A)-acetonitrile containing 0.1% formic acid(B) in a gradient mode at the flow rate of 0.3 mL·min^-1. The mass spectra were acquired with positive,high sensitivity,IDA mode. The capillary voltage was 5.5 kV,gas temperature was 550,mass scan range was m/z 200-2 000. Proteins were identified by database searching using ProteinPilot. Peptides and proteins were considered identified if they could be established at greater than 95% probability,and all matched spectra were confirmed by manual inspection. The accepted false discovery rates(FDR) for peptides and proteins were less than or equal to 1%. Results: 29 kinds of HSV proteins,most of which were capsid proteins and envelope proteins,were identified by searching HSV protein database. Two kinds of host cell proteins were identified in the protein database of Chlorocebus. One kind of Feline sarcoma virus protein was identified in the whole virus protein database. Conclusion: LC-MS has high sensitivity and resolution,which can be used for the quality research of gene therapy products.

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