Analysis of Intact Proteins in Complex Mixtures

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Our goal is to develop an effective work flow for analysis of intact proteins in a complex mixture using the LC-LTQ-Orbitrap XL. Intact protein analysis makes the entire sequence available for characterization, which allows for the identification of isoforms and post translational modifications. We focus on developing a method for top-down proteomics using a high-resolution, high mass accuracy analyzer coupled with bioinformatics tools. The complex mixtures are fractionated using 1-dimensional reversed-phase chromatography and basic reversed- phase, and open tubular electrophoresis. The analysis of intact proteins requires various fragmentation methods such as collisional induced dissociation, high energy collisional dissociation, and electron transfer dissociation. This overall method enables us to analyze intact proteins, providing a better understanding of protein expression levels and post transitional modification information. We have used standard proteins to optimize HPLC conditions and to compare three methods for ion activation and dissociation. Furthermore, we have extended the method to analyze low mass proteins in MCF7 cytosol and in E. coli lysate as a model complex mixture. We have applied this strategy to identify and characterize proteins from extracellular vesicles (EVs) shed by murine myeloid-derived suppressor cells (MDSC). MDSCs suppress both innate and adaptive immune responses to tumor growth and prevent effective immunotherapy. Recently some of the intercellular immunomodulatory effects of MDSC have been shown to be propagated by EVs. Top-down analysis of intact proteins from these EVs was undertaken to identify low mass protein cargo, and to characterize post-translational modifications.