An Integrated Strategy for Mass Spectrometry-Based Multiomics Analysis of Single Cells

Single-cell-based
genomics and transcriptomics analysis have revealed
substantial cellular heterogeneity among seemingly identical cells.
Knowledge of the cellular heterogeneity at multiomics levels is vital
for a better understanding of tumor metastasis and drug resistance,
stem cell differentiation, and embryonic development. However, unlike
genomics and transcriptomics studies, single-cell characterization
of metabolites, proteins, and post-translational modifications at
the omics level remains challenging due to the lack of amplification
methods and the wide diversity of these biomolecules. Therefore, new
tools that are capable of investigating these unamplifiable “omes”
from the same single cells are in high demand. In this work, a microwell
chip was prepared and the internal surface was modified for hydrophilic
interaction liquid chromatography-based tandem extraction of metabolites
and proteins and subsequent protein digestion. Next, direct electrospray
ionization mass spectrometry was adopted for single-cell metabolome
identification, and a data-independent acquisition-mass spectrometry
approach was established for simultaneous proteome profiling and phosphoproteome
analysis without phosphopeptide enrichment. This integrated strategy
resulted in 132 putatively annotated compounds, more than 1200 proteins,
and the first large-scale phosphorylation data set from single-cell
analysis. Application of this strategy in chemical perturbation studies
provides a multiomics view of cellular changes, demonstrating its
capability for more comprehensive investigation of cellular heterogeneity.