建立一种分离低丰度和高丰度血浆蛋白的优化方法

Henian Yang, Guijie Wang, Tiantian Zhang, J. Beattie, Shaobo Zhou
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引用次数: 2

摘要

该研究测试了一种最佳分离血浆中低丰度和高丰度蛋白质的方法的效率和再现性。首先,研究了三种分离和浓缩洗脱(E:低丰度)或结合(B:高丰度)蛋白质的方法:TCA蛋白质沉淀、ReadyPrep™ 2-D清洁套件和Vivaspin Turbo 4,5kDa超滤装置。其次,通过E和B蛋白的定量来评估Seppro柱或蛋白质提取物白蛋白/IgG柱的效率和再现性。第三,通过测量剩余洗脱体积和最终蛋白质浓度来评估含有25%或10%甘油的两种洗脱缓冲液用于洗脱结合蛋白的效率。与用TCA蛋白沉淀和ReadyPrep处理的样品相比™ 2-D清洁试剂盒,通过Vivaspin4,5kDa超滤装置浓缩的E和B蛋白在1-D和2-D凝胶中都被很好地分离。样品处理和再生15个周期后,Seppro柱中丰富蛋白质的消耗效率降低,E/(B+E)×100%的平均比值为37±11(%),样品再现性差,变异系数高(CV=30%)。然而,当使用蛋白质提取物白蛋白/IgG柱时,E/(B+E)×100%的比率为43±3.1%(n=6),其CV为7.1%,显示出良好的再现性。此外,含有10%(w/v)甘油的洗脱缓冲液提高了从蛋白质提取物白蛋白/IgG柱中洗脱B蛋白的速率,并且当用Vivaspin Turbo 4,5kDa超滤装置浓缩时,也可以获得用于2-D凝胶测定的适当蛋白质浓度(3.5µg/µl)。总之,当使用含有10%(w/v)甘油的洗脱缓冲液时,蛋白质提取物白蛋白/IgG柱显示出制备低丰度和高丰度血浆蛋白的良好再现性。制备低/高丰度血浆蛋白的优化方法是,当血浆通过蛋白质提取物白蛋白/IgG去除柱洗脱时,用含有10%甘油的洗脱缓冲液进一步洗涤该柱。分别含有低丰度和高丰度血浆蛋白的第一次和第二次洗脱使用Vivaspin®Turbo 4,5kDa超滤装置进一步浓缩,用于1或2-D凝胶电泳。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Establishing an optimized method for the separation of low and high abundance blood plasma proteins
The study tested the efficiency and reproducibility of a method for optimal separation of low and high abundant proteins in blood plasma. Firstly, three methods for the separation and concentration of eluted (E: low abundance), or bound (B: high abundance) proteins were investigated: TCA protein precipitation, the ReadyPrep™ 2-D cleanup Kit and Vivaspin Turbo 4, 5 kDa ultrafiltration units. Secondly, the efficiency and reproducibility of a Seppro column or a ProteoExtract Albumin/IgG column were assessed by quantification of E and B proteins. Thirdly, the efficiency of two elution buffers, containing either 25% or 10% glycerol for elution of the bound protein, was assessed by measuring the remaining eluted volume and the final protein concentration. Compared to the samples treated with TCA protein precipitation and the ReadyPrep™ 2-D cleanup Kit, the E and B proteins concentrated by the Vivaspin4, 5 kDa ultrafiltration unit were separated well in both 1-D and 2-D gels. The depletion efficiency of abundant protein in the Seppro column was reduced after 15 cycles of sample processing and regeneration and the average ratio of E/(B + E) × 100% was 37 ± 11(%) with a poor sample reproducibility as shown by a high coefficient of variation (CV = 30%). However, when the ProteoExtract Albumin/IgG column was used, the ratio of E/(B + E) × 100% was 43 ± 3.1% (n = 6) and its CV was 7.1%, showing good reproducibility. Furthermore, the elution buffer containing 10% (w/v) glycerol increased the rate of B protein elution from the ProteoExtract Albumin/IgG column, and an appropriate protein concentration (3.5 µg/µl) for a 2-D gel assay could also be obtained when it was concentrated with Vivaspin Turbo 4, 5 kDa ultrafiltration unit. In conclusion, the ProteoExtract Albumin/IgG column shows good reproducibility of preparation of low and high abundance blood plasma proteins when using the elution buffer containing 10% (w/v) glycerol. The optimized method of preparation of low/high abundance plasma proteins was when plasma was eluted through a ProteoExtract Albumin/IgG removal column, the column was further washed with elution buffer containing 10% glycerol. The first and second elution containing the low and high abundance plasma proteins, respectively, were further concentrated using Vivaspin® Turbo 4, 5 kDa ultrafiltration units for 1 or 2-D gel electrophoresis.
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