两种样品制备方法对血清蛋白质双向凝胶电泳分离效果的影响

Two-dimensional polyacrylamide gel electrophoresis-based serum protein separation:comparison of two sample preparation methods

目的评价两种样品制备方法对血清蛋白质双向凝胶电泳(2-DE)分离效果的影响,建立高分辨率、高重复性的血清2-DE图谱,为鉴定疾病相关血清蛋白质奠定基础。方法分别用热SDS法和直接溶解法处理大肠癌血清样品,采用固相pH梯度双向凝胶电泳技术分离总蛋白质,图像软件分析后,对其中3个差异蛋白质点行基质辅助激光解吸电离/飞行时间质谱鉴定。结果应用热SDS法处理血清总蛋白质进行双向电泳,可获得分辨率高、重复性好的人血清双向电泳图谱。对直接溶解和热SDS法处理的蛋白样品进行3次重复性检测,凝胶的平均蛋白质点数为675±46和702±49,平均匹配点数为573±42和623±52,匹配率为85.3%和89.6%,分析3块不同胶间蛋白质点在IEF方向的位置偏差为(0.85±0.30)mm和(0.81±0.28)mm,在SDS-PAGE方向上的偏差为(1.02±0.18)mm和(0.97±0.12)mm。热SDS处理的2-DE胶蛋白质点质谱可获得高质量质谱图,并可以检测到相对低丰度的血清蛋白质。结论热SDS法是一种更有效的血清蛋白质样品制备方法,我们利用热SDS法处理血清样品建立了分辨率较高且重复性好的人血清蛋白质双向凝胶电泳图谱。

Objective To evaluate the effects of two sample preparation methods on two-dimensional polyacrylamide gel electrophoresis （2-DE）-based serum protein separation, and produce high-resolution and reproducible 2-DE images for identifying disease-related serum protein. Methods Direct sohibilization and hot SDS methods were used separately to extract and handle the total proteins of serum samples from patients with colorectal carcinoma. Immobilized pH gradient 2-DE was used to separate the total proteins. After image analysis of silver-stained 2-D gels, 3 differential protein spots were identified by matrix-assisted laser desorption/time-of-flight mass spectrometry. Results The total proteins treated with hot SDS method were used to perform 2-DE. 2-DE patterns with high resolution and reproducibility were obtained for human serum samples. 2-DE was performed 3 times for the samples treated by direct solubilization and hot SDS methods, respectively, resulting in the average number of spots of 675±46 and 702±49, respectively. The average matching protein spots were 573±42 and 623± 52, with average matching rate of 85.3% and 89.6%, respectively. The average position deviation of matched spots in different gels was 0.85±0.30 mm and 0.81±0.28 mm in IEF direction, and 1.02±0.18 mm and 0.97±0.12 mm in SDS-PAGE direction. Mass spectrometry of the 2-D gels treated with hot SDS method generated high-quality mass spectra, and the sample preparation method allowed detection of relatively low abundance protein. Conclusion Hot SDS method is more effective for human serum protein sample preparation and well-resolved, reproducible 2-DE profiles of human serum have been established in this study.