SUBCELLULAR MASS SPECTROMETRY PROTEOMICS FOR CELL DIFFERENTIATION

Abstract

Cell–cell junctions are central sites of adhesion, signaling, and polarity establishment; however, they have remained inaccessible to proteome-wide analysis as discrete analytical units. This dissertation focuses on the membrane proteome and the adjacent subcellular to single-cell proteome at cell-cell junctions to explore the potential roles of proteins; particularly those involved in metabolite regulation or transport, given that metabolites are closely associated with cell fate determination during early embryonic development. This work extends from characterizing membrane proteome heterogeneity at distinct spatial locations within early embryonic cells at the subcellular level to investigating near single-cell proteome heterogeneity, with the goal of understanding cell fate determination. In addition, smaller human myeloid cells were studied to identify potential proteomic heterogeneity and their corresponding cellular functions. To achieve these objectives, advanced LC-MS and CE-MS-based proteomic methods were developed and evaluated to study subcellular and single-cell proteome heterogeneity, spanning from the Xenopus (frog) model to human cells. Collectively, this dissertation provides essential insights into protein regulation during cell differentiation and contributes to a deeper understanding of cellular heterogeneity in early embryonic development.