Lesur Antoine, Ancheva Lina, Kim Yeoun Jin, Berchem Guy, van Oostrum Jan, Domon Bruno
Luxembourg Clinical Proteomics Center (LCP), CRP-Santé, Strassen, Luxembourg.
Laboratory of Experimental Hemato-Oncology, CRP-Santé, Strassen, Luxembourg.
Proteomics Clin Appl. 2015 Aug;9(7-8):695-705. doi: 10.1002/prca.201400158. Epub 2015 Mar 30.
We report an immunocapture strategy to extract proteins known to harbor driver mutations for a defined cancer type before the simultaneous assessment of their mutational status by MS. Such a method bypasses the sensitivity and selectivity issues encountered during the analysis of unfractionated complex biological samples.
Fast LC separations using short nanobore columns hyphenated with a high-resolution quadrupole-orbitrap mass spectrometer have been devised to take advantage of fast MS cycle times in conjunction with sharp chromatographic peak widths to accelerate the sample analysis throughput. Such an analytical platform is well suited to analyze simple protein mixtures obtained after immunoaffinity enrichment.
After establishing the technical performance of the platform, the method was applied to the quantitative profiling of cellular Ras and EGFR protein isoforms, as well as serum amyloid A isoforms in plasma.
Immunoaffinity purification combined with fast LC-MS detection for the detection of driver mutations in tissue and tumor biomarkers in plasma samples can assist clinicians to select an optimal therapeutic intervention for patients.
我们报告了一种免疫捕获策略，用于在通过质谱同时评估已知携带特定癌症类型驱动突变的蛋白质的突变状态之前，提取这些蛋白质。这种方法绕过了在分析未分级的复杂生物样品过程中遇到的灵敏度和选择性问题。
已设计出使用短纳流柱与高分辨率四极杆 - 轨道阱质谱仪联用的快速液相色谱分离方法，以利用快速质谱循环时间和尖锐的色谱峰宽来加速样品分析通量。这样的分析平台非常适合分析免疫亲和富集后获得的简单蛋白质混合物。
在确定了该平台的技术性能后，该方法被应用于细胞Ras和EGFR蛋白异构体以及血浆中血清淀粉样蛋白A异构体的定量分析。
免疫亲和纯化结合快速液相色谱 - 质谱检测用于检测组织中的驱动突变和血浆样品中的肿瘤生物标志物，可协助临床医生为患者选择最佳治疗干预措施。
