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首页> 美国卫生研究院文献>Molecular Cellular Proteomics : MCP >Enhanced Sensitivity for Selected Reaction Monitoring Mass Spectrometry-based Targeted Proteomics Using a Dual Stage Electrodynamic Ion Funnel Interface
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Enhanced Sensitivity for Selected Reaction Monitoring Mass Spectrometry-based Targeted Proteomics Using a Dual Stage Electrodynamic Ion Funnel Interface

机译：使用双级电动离子漏斗接口提高对基于质谱的选定反应监测的目标蛋白质组学的灵敏度

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Selected reaction monitoring mass spectrometry (SRM-MS) is playing an increasing role in quantitative proteomics and biomarker discovery studies as a method for high throughput candidate quantification and verification. Although SRM-MS offers advantages in sensitivity and quantification compared with other MS-based techniques, current SRM technologies are still challenged by detection and quantification of low abundance proteins (e.g. present at ∼10 ng/ml or lower levels in blood plasma). Here we report enhanced detection sensitivity and reproducibility for SRM-based targeted proteomics by coupling a nanospray ionization multicapillary inlet/dual electrodynamic ion funnel interface to a commercial triple quadrupole mass spectrometer. Because of the increased efficiency in ion transmission, significant enhancements in overall signal intensities and improved limits of detection were observed with the new interface compared with the original interface for SRM measurements of tryptic peptides from proteins spiked into non-depleted mouse plasma over a range of concentrations. Overall, average SRM peak intensities were increased by ∼70-fold. The average level of detection for peptides also improved by ∼10-fold with notably improved reproducibility of peptide measurements as indicated by the reduced coefficients of variance. The ability to detect proteins ranging from 40 to 80 ng/ml within mouse plasma was demonstrated for all spiked proteins without the application of front-end immunoaffinity depletion and fractionation. This significant improvement in detection sensitivity for low abundance proteins in complex matrices is expected to enhance a broad range of SRM-MS applications including targeted protein and metabolite validation.
机译：选定的反应监测质谱（SRM-MS）在定量蛋白质组学和生物标志物发现研究中正扮演着越来越重要的角色，作为一种高通量候选物定量和验证方法。尽管与其他基于MS的技术相比，SRM-MS在灵敏度和定量方面均具有优势，但当前的SRM技术仍然面临着检测和定量低丰度蛋白（例如血浆中〜10 ng / ml或更低水平）的挑战。在这里，我们报告了通过将纳米喷雾电离多毛细管入口/双电动离子漏斗接口与商用三重四极杆质谱仪耦合，增强了基于SRM的靶向蛋白质组学的检测灵敏度和可重复性。由于离子传输效率的提高，与原始接口相比，新接口可观察到整个信号强度显着增强，检测限得到改善，而原始接口用于SRM测量胰蛋白酶肽的SRM，该蛋白是在一定范围内掺入非耗尽小鼠血浆的蛋白质的浓度。总体而言，平均SRM峰强度增加了约70倍。肽的平均检测水平也提高了约10倍，肽测量的重现性显着提高，如方差系数降低所表明。对于所有加标的蛋白质，都证明了在小鼠血浆中检测蛋白质范围为40至80 ng / ml的能力，而无需应用前端免疫亲和力消耗和分级分离。对于复杂基质中低丰度蛋白质的检测灵敏度的这种显着提高，有望增强SRM-MS的广泛应用，包括目标蛋白质和代谢物验证。

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