Patrie Steve M, Ferguson Jonathan T, Robinson Dana E, Whipple Dave, Rother Michael, Metcalf William W, Kelleher Neil L
Department of Chemistry, University of Illinois, Urbana, 61801, USA.
Mol Cell Proteomics. 2006 Jan;5(1):14-25. doi: 10.1074/mcp.M500219-MCP200.
A fragmentation geometry based upon axial acceleration of m/z-selected protein ions into a linear octopole ion trap allowed simultaneous production and external accumulation of fragment ions prior to m/z measurement in a FT mass spectrometer. Improved dynamic range resulting from this octopole collisionally activated dissociation resulted in a 2.5x increase in experimental throughput and a 2x increase in fragment ion matches to gene products identified and characterized in the top down fashion. The acceleration voltage for optimal fragmentation has a m/z and mass dependence, knowledge of which facilitated an automated platform for top down MS/MS on a quadrupole FT hybrid mass spectrometer. Controlled by improved software for data acquisition (e.g. using dynamic exclusion of previously identified species), automated octopole collisionally activated dissociation of samples fractionated using chromatofocusing and reversed-phase liquid chromatography achieved a significant increase in protein identification rate versus previous benchmarks. Also a batch analysis version of ProSight PTM facilitated probability-based identification of intact proteins obtained in a higher throughput fashion. In total, 101 unique proteins (5-59 kDa) were identified from whole cell lysates of Methanosarcina acetivorans grown anaerobically, including the characterization of several mispredicted start sites and biologically relevant mass discrepancies.
基于将选定质荷比（m/z）的蛋白质离子轴向加速进入线性八极杆离子阱的碎裂几何结构，使得在傅里叶变换（FT）质谱仪进行m/z测量之前，能够同时产生和外部累积碎片离子。这种八极杆碰撞激活解离所带来的动态范围的改善，使实验通量提高了2.5倍，并且与以自上而下方式鉴定和表征的基因产物的碎片离子匹配数增加了2倍。用于最佳碎裂的加速电压具有质荷比和质量依赖性，了解这一点有助于在四极杆FT混合质谱仪上构建一个用于自上而下质谱/质谱分析的自动化平台。通过改进的数据采集软件（例如使用对先前鉴定物种的动态排除）进行控制，对使用色谱聚焦和反相液相色谱分离的样品进行自动化八极杆碰撞激活解离，与先前的基准相比，蛋白质鉴定率有了显著提高。此外，ProSight PTM的批处理分析版本有助于以更高通量的方式对完整蛋白质进行基于概率的鉴定。总共从厌氧生长的嗜乙酸甲烷八叠球菌的全细胞裂解物中鉴定出101种独特蛋白质（5 - 59 kDa），包括对几个错误预测的起始位点和生物学相关质量差异的表征。
