National Center for Quantitative Biology of Complex Systems

项目总结--研发2 在一次MS分析中并行处理多个样品--即样品多路传输--已成为一种 将基于MS的系统生物学方法应用于转化医学的首选方法 拥有众多的关键优势。第一，单质谱分析中样品的并行处理 减少任何一个样品所需的材料量。第二，多路传输减少了样品的准备 和仪器分析时间要求。第三，高度复杂的分析有助于收集生物 复制数据。第四，将样品合并到单一分析中的能力允许更少的MS实验，这 反过来，允许在所有条件下有更大的数据重叠。在这个研发中，我们将开发和应用一个新的类 用于蛋白质和蛋白质翻译后修饰(PTMS)定量的同位素标记物。具体来说，标签 将建立在胺反应的N，N-二烷基氨基酸支架上，该支架与电子兼容 转移解离(ETD)。这一目标的最终目标是确定如何最有效地结合 结合ETD优势的多重量化在不稳定的PTM鉴定中的效率。第二，我们 将开发和应用化学标签，用于数据独立采集(DIA)的多路定量。至 迄今为止，只有代谢标记成功地对DIA进行了多重分析。在这里，我们将继续 开发我们的基于质量缺陷的化学标签，胺反应性二甲基嘧啶鸟氨酸(DiPyrO)标签，以 促进多路复用的定量DIA应用。这些标签将提供多达10个DIA量化，带来 这项新兴技术的多路传输能力。第三，我们将改进等压标签的MS硬件 技术特别是，我们将使用气相离子/离子反应来提纯目标分析物并将 污染物，从而提高了等压标记的定量准确性。为了提高精确度，我们将 利用红外线光激活，完全释放所有报告标签，从而增强信号。

PROJECT SUMMARY – TR&D 2 Processing multiple samples in parallel in a single MS analysis – i.e., sample multiplexing – has become a preferred method for the application of MS-based systems biology approaches to translational medicine thanks to numerous key advantages. First, parallel processing of samples in a single mass spectrometric analysis reduces the amount of material required from any one sample. Second, multiplexing reduces sample preparation and instrument analysis time requirements. Third, highly plexed analyses facilitate collection of biological replicate data. Fourth, the ability to combine samples into a single analysis permits fewer MS experiments, which in turn allows for greater data overlap across all conditions. In this TR&D, we will develop and apply a new class of isobaric tags for protein and protein post-translational modifications (PTMs) quantitation. Specifically, the tags will be built upon an amine-reactive N,N-dialkylated amino acid-based scaffold that is compatible with electron transfer dissociation (ETD). The ultimate goal of this aim is to determine how to most effectively combine the efficiencies of multiplexed quantification with the advantages of ETD for labile PTM identification. Second, we will develop and apply chemical tags for multiplexed quantification with data independent acquisition (DIA). To date, only metabolic labels have been successful in multiplexing analysis for DIA. Here we will continue to develop our mass defect-based chemical tag, amine-reactive dimethyl pyrimidinyl ornithine (DiPyrO) tags, to facilitate multiplexed quantitative DIA applications. These tags will offer up to 10-plex DIA quantification, bringing multiplexed capacity for this emergent technology. Third, we will improve MS hardware for isobaric tag technology. In particular, we will use gas-phase ion/ion reactions to purify the target analyte and remove contaminants, thereby improving the quantitative accuracy of isobaric tagging. And, to improve precision, we will leverage infrared photo-activation to completely release all reporter tags, thereby boosting signal.