Project Summary/Abstract: As the single-cell transcriptomics analysis becomes more accessible, individual cells can now be classified individually, allowing us to understand cell heterogeneity as one of the key driving forces in neuron development and tumor evolution.  However, the presence of mRNA is not always coupled with protein expression, single-cell proteomics will provide a more precise view of the cell components and signaling, including key post-translational modification events such as phosphorylation.  Here we propose to combine several different strategies to develop a highly sensitive proteomics method to probe cell heterogeneity at single cell resolution.  Specifically, we will 1) develop chemical labeling probes to enhance peptide ionization; 2) develop an isobaric isotopologue labeling approach to quantify single mammalian cell from various cell lines; 3) develop informatics tools and statistical models to resolve cell heterogeneity at single-cell resolution with non-single-cell proteomics data.  Each of these proposed methods alone, could potentially enable us to measure the proteome of a single mammalian cell.  When combined, they will result in a highly sensitive platform that could potentially allow us to probe the proteome at the level of a single mitochondria or bacteria.  The long-term goal of this project is to elucidate the fundamental mechanisms of the origin and implications of cell heterogeneity.  This proposed research is highly relevant to public health in the fields of tumor evolution, cancer metastasis, developmental neurobiology and neurodegenerative disease.  Understanding cell heterogeneity will help us to develop more effective drug treatments to many diseases.  Therefore, the proposed research is relevant to the part of NIH's mission that fosters ? fundamental creative discoveries, innovative research strategies, and their applications as a basis for ultimately protecting and improving health?  and that ? supports research in the causes, diagnosis, prevention, and cure of human diseases.?  The proposed research is relevant to the NIGMS 2015-2020 strategic plan: ? Objective 1-1: Invest in and sustain a broad and diverse portfolio of highly meritorious research.?, ? Objective 1-2: Promote the ability of investigators to pursue new research directions, novel scientific insights and innovative ideas?,  and ? Objective 3-1: Support access to essential research resources and the development of new technologies that enable novel scientific advances.?