Proteomics on an Orbitrap benchtop mass spectrometer using all-ion fragmentation.
Proteomics on an Orbitrap benchtop mass spectrometer using all-ion fragmentation.
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DOI：
10.1074/mcp.m110.001537
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发表时间：
2010-10
期刊：
影响因子：
--
通讯作者：
Mann M
中科院分区：
文献类型：
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作者：
Geiger T;Cox J;Mann M
轨道阱质量分析仪以紧凑的形式结合了高灵敏度、高分辨率和高质量精度。在蛋白质组学应用中，它与线性离子阱（LTQ-Orbitrap）一起用于混合配置，其中线性阱四极杆（LTQ）积累，分离和片段肽离子。或者，分离的离子可以通过更高能量的碰撞解离而碎裂。最近推出的独立轨道阱分析仪（Exactive）也具有更高能量的碰撞解离单元，但不能分离离子。在这里，我们报告说，该仪器可以有效地表征蛋白质混合物的交替MS和“全离子碎片”（AIF）MS/MS扫描的方式类似于先前描述的四极杆飞行时间仪器。我们在前体和产物离子水平上应用MaxQuant软件的峰识别算法。通过高分辨率（m/z 200时为100，000）和高质量准确度，促进了碎片离子与共洗脱前体离子的归属。对于不同质量前体的有效碎片化，我们实施了具有累积MS读出的阶梯式碰撞能量程序。Exactive上的AIF在等摩尔蛋白质标准混合物中鉴定了48种蛋白质中的45种，并且在使用小型数据库时鉴定了所有蛋白质。该技术还鉴定了在高动态范围标准中具有超过100倍丰度差异的蛋白质。当应用于凝胶切片中的蛋白质鉴定时，AIF明确地表征了通过考马斯染色几乎不可见的免疫沉淀蛋白质，并相对于污染蛋白质对其进行定量。因此，在台式轨道阱仪器上的AIF是一种有吸引力的技术，用于广泛的蛋白质组学分析。
The orbitrap mass analyzer combines high sensitivity, high resolution, and high mass accuracy in a compact format. In proteomics applications, it is used in a hybrid configuration with a linear ion trap (LTQ-Orbitrap) where the linear trap quadrupole (LTQ) accumulates, isolates, and fragments peptide ions. Alternatively, isolated ions can be fragmented by higher energy collisional dissociation. A recently introduced stand-alone orbitrap analyzer (Exactive) also features a higher energy collisional dissociation cell but cannot isolate ions. Here we report that this instrument can efficiently characterize protein mixtures by alternating MS and “all-ion fragmentation” (AIF) MS/MS scans in a manner similar to that previously described for quadrupole time-of-flight instruments. We applied the peak recognition algorithms of the MaxQuant software at both the precursor and product ion levels. Assignment of fragment ions to co-eluting precursor ions was facilitated by high resolution (100,000 at m/z 200) and high mass accuracy. For efficient fragmentation of different mass precursors, we implemented a stepped collision energy procedure with cumulative MS readout. AIF on the Exactive identified 45 of 48 proteins in an equimolar protein standard mixture and all of them when using a small database. The technique also identified proteins with more than 100-fold abundance differences in a high dynamic range standard. When applied to protein identification in gel slices, AIF unambiguously characterized an immunoprecipitated protein that was barely visible by Coomassie staining and quantified it relative to contaminating proteins. AIF on a benchtop orbitrap instrument is therefore an attractive technology for a wide range of proteomics analyses.