Enhanced Sensitivity for Selected Reaction Monitoring Mass Spectrometry-based Targeted Proteomics Using a Dual Stage Electrodynamic Ion Funnel Interface
Enhanced Sensitivity for Selected Reaction Monitoring Mass Spectrometry-based Targeted Proteomics Using a Dual Stage Electrodynamic Ion Funnel Interface
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DOI：
10.1074/mcp.m000062-mcp201
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发表时间：
2011-02-01
影响因子：
7
通讯作者：
Smith, Richard D.
中科院分区：
文献类型：
--
作者：
Hossain, Mahmud;Kaleta, David T.;Smith, Richard D.
选择反应监测质谱仪(SRMMS)作为一种高通量的候选定量和验证方法，在定量蛋白质组学和生物标志物发现研究中发挥着越来越重要的作用。尽管与其他基于MS的技术相比，SRM-MS在灵敏度和定量方面具有优势，但当前的SRM技术仍然面临着低丰度蛋白质(例如，血浆中含量接近10 ng/ml或更低水平)的检测和定量的挑战。在这里，我们报告了通过将纳米螺旋电离多毛细管入口/双电动离子漏斗接口耦合到商用三重四极杆质谱仪来提高基于SRM的靶向蛋白质组的检测灵敏度和重复性。由于离子传输效率的提高，与原来的SRM接口相比，新接口在总体信号强度和检测极限方面都有了显著的增强，用于从添加到未耗尽的小鼠血浆中的蛋白质在一定浓度范围内对胰蛋白酶多肽进行SRM测量。总体而言，SRM的平均峰值强度增加了约70倍。多肽的平均检测水平也提高了近10倍，由降低的变异系数表明，多肽测量的重复性显著提高。在没有应用前端免疫亲和力耗尽和分级的情况下，所有添加的蛋白质都证明了能够检测到小鼠血浆中从40到80 ng/ml的蛋白质。这种对复杂基质中低丰度蛋白质检测灵敏度的显著提高有望加强SRM-MS的广泛应用，包括靶向蛋白质和代谢物验证。分子与细胞蛋白质组学10：10.1074/mcp.M000062-MCP201，1-9,2011年。
Selected reaction monitoring mass spectrometry (SRMMS) 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 similar to 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 similar to 70-fold. The average level of detection for peptides also improved by similar to 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. Molecular & Cellular Proteomics 10:10.1074/mcp.M000062-MCP201, 1-9, 2011.