目的: 采用液质联用（LC-MS/MS）技术分析间充质干细胞（MSCs）表面标志蛋白,探索MSCs表面标志蛋白鉴定新方法。方法: 用RIPA裂解液裂解MSCs,用BCA试剂盒定量细胞裂解液蛋白浓度,将MSCs蛋白还原烷基化后用胰蛋白酶酶切,酶切肽段进行LC-MS/MS分析。采用ACQUITY Premier Peptide BEH C18（150 mm×2.1 mm,1.7 µm）色谱柱,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0.2 mL · min-1;质谱仪采用正离子、灵敏度、数据依赖型（DDA）模式采集数据,毛细管电压2.5 kV,锥孔电压40 V,离子源温度120 ℃,扫描范围m/z 50~2 000;将采集的质谱数据导入到waters connect软件中进行搜库检索,前体离子的质量容差为0.001%,碎片离子的质量容差为0.002%,设置至少3个b/y离子匹配为肽鉴定的标准。结果: 通过DDA模式,鉴定到MSCs阳性标志蛋白CD90;进一步在DDA模式下引入Include list,鉴定到阳性标志蛋白CD73,但还未能鉴定到阳性标志蛋白CD105;阴性标志蛋白CD34、CD45、CD14、CD19、CD79a及HLA-DR未检出。结论: LC-MS/MS技术具有快速、准确及不依赖于抗体的特点,已分析出2个阳性标志蛋白,通过进一步研究和方法探索,有望用于MSCs表面标志蛋白及目标蛋白的鉴定。
Objective: To adopt liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze surface marker proteins of mesenchymal stem cells (MSCs), exploring a novel methodology for their identification. Methods: MSCs were lysed with RIPA buffer, and the protein concentration of cell lysates was quantified using a BCA kit. After reduction and alkylation of MSCs proteins, trypsin digestion was performed, and the resulting peptides were analyzed by LC-MS/MS. The chromatographic separation was carried out on An ACQUITY Premier Peptide BEH C18 column (150 mm×2.1 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 a flow rate of 0.2 mL · min-1. The mass spectrometer was operated in positive ion mode, sensitivity, data-dependent acquisition (DDA) mode, with capillary voltage set at 2.5 kV, cone voltage at 40 V, ion source temperature at 120 ℃, and a scan range of m/z 50-2 000. Proteins were identified by database searching using waters connect software. Mass tolerance was set as 0.001% for precursor ions and 0.002% for fragmentation ions. And a minimum of three b/y-ion matches were set as the criteria for peptide identification. Results: Through DDA mode, the positive marker CD90 of MSCs was identified. Further introduction of an include list under DDA mode led to the identification of the positive marker CD73, while the positive marker CD105 was not identified. Negative markers including CD34, CD45, CD14, CD19, CD79a, and HLA-DR were not detected. Conclusion: LC-MS/MS technology is rapid, accurate, and antibody-independent. It has identified two positive marker proteins. Through further research and methodological exploration, this technique can be applied to the identification of MSC surface marker proteins and target proteins.
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