National Center for Quantitative Biology of Complex Systems

点击翻译按钮获取中文摘要

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.