Project Summary/Abstract
Proteoforms are molecular forms of proteins in cells and tissues containing site-specific sequence
variations and post-translational modifications. Proteoforms effectively describe cell phenotypes
and provide important implications in disease mechanisms. Recent studies in immunobiology and
disease pathology have emphasized intact proteoform characterization without enzymatic
digestion in bottom-up approaches. Current mass-spectrometry-based top-down proteomics
technologies fall short in capturing the complex proteome in small biological samples such as
single cells. This proposal generates a suite of novel technologies for high-throughput omics-
scale single cell proteoform profiling using innovative instrumentation, bioinformatics and high-
throughput strategies. I will use human kidney cells as a model biological system in this proposal,
and the approaches are generalizable to different cell types. I have recently developed a
technology employing localized proteoform sampling coupled to single-molecule mass
spectrometry to directly image and identify intact proteoforms in tissue sections (Su et al., 2022,
Sci. Adv.). In Aim 1, I will expand the proteome detection and identification capabilities in this
technology using innovative instrumentation, sampling and data acquisition algorithms. Together
these will curate a knowledgebase serving as a proteoform library for kidney single cell proteoform
profiling. In Aim 2, I will develop a novel platform leveraged for profiling of single cells dissociated
from human kidney biopsies. I will develop a single cell preparation protocol for maximizing
proteoform detection in kidney parenchymal and immune cells. I will also develop a bioinformatics
approach tailored for proteoform identification in single cell datatype and discover proteoform
signatures that differentiate cell types. In Aim 3, I will address the limitation in rare cell profiling by
developing a series of high-throughput strategies including high-speed sampling and microarray
cell patterning. These new technologies will unravel proteoform landscapes and signatures in rare
kidney-resident innate immune cells (e.g., macrophages and dendritic cells) for the discovery of
new cell populations that can be used as therapeutic targets and diagnostic tools for inflammatory
diseases. My mentoring team consists of Dr. Neil Kelleher (mentor), a world-renowned protein
biochemist, and Dr. Satish Nadig (co-mentor), a leading expert in kidney immunobiology. The
proposed research is a substantial technological advancement in single-cell proteomics and sets
a solid foundation for the pursuit of my independent career. The proposed research also well
aligns with my long term research interest in developing enabling analytical technologies for
biomedical science community with a special interest in the human innate immune system.