PROJECT SUMMARY/ABSTRACT
Single-cell technologies have become the cornerstone of biomedical and cell biology research. Next-
generation sequencing-based technologies have enabled large-scale characterization of transcript expressions
in single cells from clinical specimens and reveal unexpected cellular heterogeneity related to pathogenesis.
However, many integrative studies have shown only low to moderate correlations between the abundance of
RNA transcripts and their corresponding proteins, the main determinants of cell phenotype. We hypothesize
mass spectrometry-based single-cell proteomics could provide direct insight on the cellular heterogeneity and
inform protein markers related to disease progression and resistance to therapy. The overall objective of this
project is to develop a high throughput single-cell proteomics (scProteomics) platform to enable the routine
analysis of >10,000 single cells at a depth of 2000 proteins in a cost-efficient way. The developed technology
will be disseminated to the research community through close collaboration with a commercial partner. We will
also apply scProteomics to interrogate the heterogeneity of both malignant plasma cell and immune cell
populations from multiple myeloma patients. We will pursue these goals through three specific aims: 1) To
establish an ultra-high throughput single-cell preparation method by coupling an enhanced multiplexing method
with high-density nested nanoPOTS chips and multi-channel droplet dispensing system; We aim to process
>2000 cells in a single microchip, and multiplex-label 36 single cells for a single LC-MS analysis; 2) To
advance the throughput, sensitivity, and quantitation accuracy of LC-MS system. A dual-column nanoLC
system and a FAIMS-based MS acquisition method will be developed to enable the analysis of >860 cells per
day with high quantitation precision; 3) To apply scProteomics to profile ~10,000 plasma and immune cells
from MM patients. We will integrate scProteomics with existing scRNA-seq data to explore tumor
heterogeneity, chimeric antigen receptor T-cells (CAR-T) markers, and the immune microenvironment in
multiple myeloma. This research is highly innovative because the proposed single-cell proteomics platform will
be the first of its kind to routinely and reliably characterize > 10,000 single cells at a throughput comparable to
single-cell transcriptomics. It is also the first scProteomics study of primary liquid tumor cells isolated from the
pathological environment, e.g. bone marrow of MM patients. Statement of Impact: Tumor heterogeneity has
indispensable implications in cancer evolution, tumoral spatial organization, and clinical treatment. Single-cell
proteomics could provide a basis to unravel these complicated relationships and to clarify the mechanisms of
cancer progression and subclone resistance to therapeutic treatments.