High Resolution Parallel Reaction Monitoring with Electron Transfer Dissociation for Middle-Down Proteomics
High Resolution Parallel Reaction Monitoring with Electron Transfer Dissociation for Middle-Down Proteomics
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DOI：
10.1021/acs.analchem.5b01542
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发表时间：
2015-08-18
影响因子：
7.4
通讯作者：
Hess, Sonja
中科院分区：
文献类型：
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作者：
Sweredoski, Michael J.;Moradian, Annie;Hess, Sonja
近年来，中下蛋白质组学已经成为一种流行的技术，用于表征和定量蛋白质，而这些蛋白质不容易接受典型的自下而上的方法。到目前为止，所有的高分辨率中下方法都是在数据依赖的采集模式下完成的，使用碰撞诱导解离或电子捕获/转移解离技术。在这里，我们探索中间向下的蛋白质组学与电子转移解离使用有针对性的采集模式，并行反应监测（PRM），在Orbitrap融合。作为高度修饰的蛋白质的一个例子，我们使用来自未处理和DMSO处理的鼠红白血病（MEL）细胞的组蛋白H3级分。我们首先确定了优化的仪器参数，以获得高的序列覆盖使用合成的标准肽。然后，我们建立了H3的N-末端尾部的+10电荷状态的甲基化和乙酰化的20种最丰富的组合的MS 1扫描和PRM扫描的组合方法。弱阳离子交换亲水相互作用色谱法用于分离N-末端H3尾，主要是通过其乙酰化，其次是通过其甲基化状态，这有助于解释结果。在高电荷离子的去卷积后，将峰注释为未处理和处理样品中的254个H3蛋白质型的最小集合。DMSO处理后，相对于MS 1水平的总体定量变化显示2、3、4和5个乙酰化相对减少，0和1个乙酰化增加。开发碎片离子图以可视化处理和未处理样品之间的特定差异。总之，这里提供的数据表明，使用PRM的电子转移解离的中间蛋白质组学是一种新的，有吸引力的方法，用于有效分析和定量的大型和高度修饰的肽。
In recent years, middle-down proteomics has emerged as a popular technique for the characterization and quantification of proteins not readily amenable to typical bottom-up approaches. So far, all high resolution middle-down approaches are done in data-dependent acquisition mode, using both collision-induced dissociation or electron capture/transfer dissociation techniques. Here, we explore middle-down proteomics with electron transfer dissociation using a targeted acquisition mode, parallel reaction monitoring (PRM), on an Orbitrap Fusion. As an example of a highly modified protein, we used histone H3 fractions from untreated and DMSO-treated Murine ErythroLeukemia (MEL) cells. We first determined optimized instrument parameters to obtain high sequence coverage using a synthetic standard peptide. We then setup a combined method of both MS1 scans and PRM scans of the 20 most abundant combinations of methylation and acetylation of the +10 charge state of the N-terminal tail of H3. Weak cation exchange hydrophilic interaction chromatography was used to separate the N-terminal H3 tail, primarily, by its acetylation and, to a secondary degree, by its methylation status, which aided in the interpretation of the results. After deconvolution of the highly charged ions, peaks were annotated to a minimum set of 254 H3 proteoforms in the untreated and treated samples. Upon DMSO treatment, global quantitation changes from the MS1 level show a relative decrease of 2, 3, 4, and 5 acetylations and an increase of 0 and 1 acetylations. A fragment ion map was developed to visualize specific differences between treated and untreated samples. Taken together, the data presented here show that middle-down proteomics with electron transfer dissociation using PRM is a novel, attractive method for the effective analysis and quantification of large and highly modified peptides.