The Impact II, a Very High-Resolution Quadrupole Time-of-Flight Instrument (QTOF) for Deep Shotgun Proteomics.
The Impact II, a Very High-Resolution Quadrupole Time-of-Flight Instrument (QTOF) for Deep Shotgun Proteomics.
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DOI：
10.1074/mcp.m114.047407
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发表时间：
2015-07
期刊：
影响因子：
--
通讯作者：
Mann M
中科院分区：
文献类型：
--
作者：
Beck S;Michalski A;Raether O;Lubeck M;Kaspar S;Goedecke N;Baessmann C;Hornburg D;Meier F;Paron I;Kulak NA;Cox J;Mann M
混合四极杆飞行时间质谱仪(QTOF)是蛋白质组学的两个主要原理之一。虽然基于简单的基本原理，但在过去的几十年中，它在可实现的分辨率、质量精度和动态范围方面有了很大的发展。QTOF仪器的Bruker Impact平台利用了这些发展，在这里我们开发和评估了用于鸟枪式蛋白质组学应用的Impact II。采用我们的加热液相色谱系统可以得到非常窄的多肽洗脱峰。Impact II配备了一个新的碰撞池，具有轴向和径向离子喷射，在高串联MS频率下离子引出增加了一倍多。与以前的型号相比，新的反射器和探测器的分辨率提高了80%，即在m/z 1222时提高到40,000。我们分析了来自进口毛细管的离子电流，发现非常高的透过率(>；80%)到碰撞电池。模拟和测量表明，60%的物质转移到了飞行管内。我们将MaxQuant用于QTOF数据，将绝对平均质量偏差改进为优于1.45ppm。在90分钟的梯度中，一次HeLa消化可以鉴定出超过4800种蛋白质。该工作流程实现了高技术重复性(R2>；0.99)，并在复杂混合物的加压实验中准确确定折叠变化。利用非标记定量技术，我们对二倍体酵母的单倍体进行了快速定量，并表征了来自不同组织的小鼠细胞系的总体蛋白质组差异。最后，经过高pH反相分离后，我们在HeLa多肽混合物的三次测量中鉴定出9515种蛋白质，在小脑的一次测量中鉴定出11,257种蛋白质--这是迄今为止QTOF仪器报告的最高蛋白质组覆盖率。
Hybrid quadrupole time-of-flight (QTOF) mass spectrometry is one of the two major principles used in proteomics. Although based on simple fundamentals, it has over the last decades greatly evolved in terms of achievable resolution, mass accuracy, and dynamic range. The Bruker impact platform of QTOF instruments takes advantage of these developments and here we develop and evaluate the impact II for shotgun proteomics applications. Adaption of our heated liquid chromatography system achieved very narrow peptide elution peaks. The impact II is equipped with a new collision cell with both axial and radial ion ejection, more than doubling ion extraction at high tandem MS frequencies. The new reflectron and detector improve resolving power compared with the previous model up to 80%, i.e. to 40,000 at m/z 1222. We analyzed the ion current from the inlet capillary and found very high transmission (>80%) up to the collision cell. Simulation and measurement indicated 60% transfer into the flight tube. We adapted MaxQuant for QTOF data, improving absolute average mass deviations to better than 1.45 ppm. More than 4800 proteins can be identified in a single run of HeLa digest in a 90 min gradient. The workflow achieved high technical reproducibility (R2 > 0.99) and accurate fold change determination in spike-in experiments in complex mixtures. Using label-free quantification we rapidly quantified haploid against diploid yeast and characterized overall proteome differences in mouse cell lines originating from different tissues. Finally, after high pH reversed-phase fractionation we identified 9515 proteins in a triplicate measurement of HeLa peptide mixture and 11,257 proteins in single measurements of cerebellum—the highest proteome coverage reported with a QTOF instrument so far.