Top down mass spectrometry of <60-kDa proteins from Methanosarcina acetivorans using quadrupole FTMS with automated octopole collisionally activated dissociation
Top down mass spectrometry of <60-kDa proteins from Methanosarcina acetivorans using quadrupole FTMS with automated octopole collisionally activated dissociation
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DOI：
10.1074/mcp.m500219-mcp200
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发表时间：
2006-01-01
影响因子：
7
通讯作者：
Kelleher, NL
中科院分区：
文献类型：
--
作者：
Patrie, SM;Ferguson, JT;Kelleher, NL
基于m/z选择的蛋白质离子轴向加速进入线性八极离子阱的碎片几何形状允许在FT质谱仪中进行m/z测量之前同时产生和外部积累碎片离子。由这种八极碰撞激活解离产生的改进的动态范围导致实验通量增加2.5倍，并且与以自上而下的方式鉴定和表征的基因产物的片段离子匹配增加2倍。用于最佳碎裂的加速电压具有m/z和质量依赖性，其知识促进了在四极杆FT混合质谱仪上用于自顶向下MS/MS的自动化平台。通过改进的数据采集软件进行控制（例如，G.使用先前鉴定的种类的动态排除），使用色谱聚焦和反相液相色谱法分级分离的样品的自动八极碰撞活化解离与先前的基准相比实现了蛋白质鉴定率的显著增加。此外，ProSight PTM的批分析版本促进了以更高通量方式获得的完整蛋白质的基于概率的鉴定。总之，从厌氧生长的乙酸甲烷八叠球菌的全细胞裂解物中鉴定出101种独特的蛋白质（5 - 59 kDa），包括几个错误预测的起始位点和生物学相关质量差异的表征。
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 2.5 x increase in experimental throughput and a 2 x 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.