Structural Mapping Protein-Surface Interface by Proteomics

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The ability to study proteins and membrane proteins in a small number of cells is critical to investigate stem-cell development and the cause of cancer. Proteome is the entire protein complement of a biological system. Proteomic study of a small number of stem cells and cancer initiating cells enables comprehensive characterization of protein functions in complex biological events that these cells participate in. The obtained knowledge can provide molecular guidance for disease treatment and prevention.Currently it is impossible to carry out membrane proteomics from fewer than 1000 cells, which is needed to address the above mentioned biological questions. One reason is the severe sample loss occurs in proteomic analysis of trace proteins (i.e. microproteomics). Aspecific adsorption of proteins not only hampers microproteomics, but also impacts a number of other research fields including biosensing, biomedicine, biodefense, as well as food, marine, and textile industries. Protein surface interactions are complex. A majority of proteins in the proteome have no structural information on how they interact with different solid surfaces; therefore, there is no effective strategy to prevent proteome-wise protein adsorption.CFI has awarded us a nanoflow liquid chromatography (LC) and accurate Fourier transform mass spectrometer (MS) coupled system for us to develop novel methodologies to investigate molecular structures at the protein-surface interface, and to analyze membrane proteome from a few stem cells. In the proposed research, we will use Chinese hamster ovary cells as a model and the awarded LC-MS system as the detection means to explore different chaotropes, surfactants, enzymes, and proteolytic methods for proteomic characterization of sub-microgram of protein adsorbed in regular sample vials. By tagging individual amino acids, we will probe the atomic interfacial structure for hundreds if not thousands of proteins. By modulating such interaction, we hope to achieve the sensitive analysis of proteome and membrane proteome from a few cells.This study will enable us to utilize developed methods to investigate the cell population heterogeneity not only in stem cells that prohibits its clinical translation, but also in cancer tissues that causes fractional kills in current therapies and frequent relapse. The discovered biomarkers of different subtypes of stem cells will facilitate the production of high quality and quantity stem cells needed in clinics, and the discovered druggable targets will enable complete eradication of cancer. The developed methods will also benefit other sensitive proteomic studies such as early diagnosis and prevention of diseases. The strategies to control protein aspecific adsorption will shed light on other research fields mentioned above, and the students trained in our program will help the research and application in these fields in the future.