National Center for Quantitative Biology of Complex Systems

项目概要- TR&D 2 在单个MS分析中并行处理多个样品-即，样本多路复用-已成为 基于MS的系统生物学方法应用于转化医学的首选方法， 许多关键优势。首先，在单个质谱分析中并行处理样品 减少了任何一个样品所需的材料量。第二，多路复用减少了样品制备 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信息。 复制数据。第四，将联合收割机样品组合成单一分析的能力允许更少的MS实验， 这又允许在所有条件下有更大的数据重叠。在本TR&D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（PTM）定量的同量异位素标签。具体来说，标签 将建立在与电子相容的胺反应性N，N-二烷基化氨基酸基支架上， 转移解离（ETD）。该目标的最终目标是确定如何最有效地将联合收割机 多重定量的效率与ETD用于不稳定PTM鉴定的优点。二是 将开发和应用化学标签，用于数据独立采集（DIA）的多重定量。到 迄今为止，只有代谢标记物在DIA的多重分析中取得了成功。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基鸟氨酸（DiPyrO）标签， 便于多路定量DIA应用。这些标签将提供高达10重的DIA定量， 多路复用的能力。第三，我们将改进用于等压标签的MS硬件 技术.特别地，我们将使用气相离子/离子反应来纯化目标分析物并去除 污染物，从而提高同量异位素标记的定量准确性。为了提高精度，我们将 利用红外光活化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.