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首页> 外文期刊>Analytical and bioanalytical chemistry >BRAF protein immunoprecipitation, elution, and digestion from cell extract using a microfluidic mixer for mutant BRAF protein quantification by mass spectrometry
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BRAF protein immunoprecipitation, elution, and digestion from cell extract using a microfluidic mixer for mutant BRAF protein quantification by mass spectrometry

机译：使用微流体混合器通过质谱法测量突变体BRAF蛋白的细胞提取物的BRAF蛋白免疫沉淀，洗脱和消化

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摘要

This study utilized a microfluidic mixer for the sample pretreatment of cell extracts for target protein quantification by mass spectrometers, including protein immunoprecipitation and protein enzymatic digestion. The time of sample pretreatment was reduced and thus the throughput of quantitative mutant proteins was increased by using the proposed method. Whole cell lysates of the cancer cell line HT-29 with gene mutations were used as the sample. The target protein BRAF was immunoprecipitated using magnetic beads in a pneumatic micromixer. Purified protein was then eluted and digested by trypsin in another two micromixers to yield peptide fragments in the solution. Using stable isotope-labeled standard as the internal control, wild-type and mutant BRAF proteins were quantified using mass spectrometry, which could be used for cancer screening. Compared with conventional methods in which protein immunoprecipitation lasts overnight, the micromixer procedure takes only 1h, likely improving the throughput of mutant BRAF protein quantification by mass spectrometry.
机译：该研究利用微流体混合器用于通过质谱仪进行靶蛋白质定量的细胞提取物的样品预处理，包括蛋白质免疫沉淀和蛋白质酶消化。通过使用所提出的方法，降低了样品预处理的时间，从而增加了定量突变蛋白的产量。使用具有基因突变的癌细胞系HT-29的全细胞裂解物作为样品。使用气动微混合物中的磁珠免疫沉淀靶蛋白BRAF。然后通过另外两个微混合物中的胰蛋白酶洗脱并消化纯化的蛋白质，以产生溶液中的肽片段。使用稳定的同位素标记标准作为内部控制，使用质谱法定量野生型和突变蛋白酶蛋白，其可用于癌症筛选。与蛋白质免疫沉淀持续过夜的常规方法相比，微混合器程序仅需要1小时，可能改善质谱法量化的突变BRAF蛋白质量化的产量。

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