||5UH3CA256959-04 Interpret this number
||Battelle Pacific Northwest Laboratories
||Spatially Resolved Characterization of Proteoforms for Functional Proteomics
Differentiated cells have distinctive patterns of epigenetic marks including various post-translational
modifications (PTMs) on histones that may work in concert to control transcriptional programs. Since epigenetic
marks are often altered following exposure to environmental toxins and play multiple roles in disease
pathogenesis, the ability to measure histones in a tissue and cell context is a major analytical objective and
challenge. Mass spectrometry (MS) based proteomics is a powerful tool for characterizing histone alterations in
multiplexed and non-targeted fashion. However, conventional bottom-up (i.e. peptide-level) MS cannot provide
complete characterization of the stoichiometry and combinations of multiple PTMs, and other combinatorial
sources of variation, that collectively make up any single gene's set of proteoforms (i.e. functional units of a
proteome). Top-down (i.e. proteoform-level) MS addresses this challenge by omitting the proteolysis and thus
allowing access to the functional proteoforms. However, top-down MS suffers from low sensitivity and dynamic
range due to challenges in separation and detection of large and low-abundance proteins and laborious
purification steps required to achive high proteome coverage. This severely limits our ability to analyze small
samples and employ top-down MS to generate proteoform-aware images of tissues required for a deeper
understanding of human organ functioning in health and disease. We have recently developed nanodroplet
sample preparation (nanoPOTS) for highly sensitive bottom-up proteomics and extended this approach to tissue
imaging with 100 µm spatial resolution. Herein, we propose to develop and deploy nanoPOTS-based top-down
MS to enable characterization of proteoforms in tissue sections with near single cell resolution. To increase the
resolution from thousands of cells to near single cell, we will employ advanced MS imaging (MSI) approaches.
MSI data will be cross-referenced with global proteomics data obtained via microscale top-down MS of
microdissected tissue regions. The UG3 phase efforts will be focused on histones and kidney as a development
platform and leverage a unique combination of microscale top-down LCMS, MSI and novel image processing
and visualization tools. In the UH3 phase, we will construct comprehensive proteoform-specific maps of multiple
tissue types and facilitate multimodal molecular mapping of specific functional units of the kidney by leveraging
the HubMAP Consortium ongoing efforts. Successful completion of this research will allow for comprehensive
characterization of the full spectrum of proteoforms in tissues and cells thus addressing an important and under-
studied area of biology and critical gap in HuBMAP efforts.
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