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

Abstract

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.

Optimized data-independent acquisition approach for proteomic analysis at single-cell level

“…Furthermore, identification of PTMs at nano-gram/single-cell level without any PTM enrichment was achieved by directly searching the nano-gram level of global DIA data against pre-generated PTM libraries. More PTM sites may be identified if the PTM libraries constructed by PTM enrichments from the same samples are used to search the global proteomic data [ 39 ]. Additionally, we were able to detect the cellular heterogeneity between PACCs and their parental MDA-MB-231 cells at single-cell level using our established workflow.…”

Proteomics in Biomarker Discovery for Tuberculosis: Current Status and Future Perspectives

“…It is important to consider that, coupled with proteomic technologies, proteomics databases, including Swiss-Prot, Entrez, and the Human Proteome Organization, also continue to contribute to the development of proteomics ( Kavallaris and Marshall, 2005 ). Moreover, in addition to other single-cell approaches ( Mauger et al, 2021 ), single-cell proteomics is emerging as a way to identify and quantify the proteins in individual cells ( Li et al, 2021 ). Although there are no high-throughput proteomic “sequencers” available as yet, it is believed that this technology will contribute to the discovery of new aspects of protein and cell biology in the future.…”

Standard-free single magnetic bead evaluation: a stable nanoplatform for prostate disease differentiation

“…Absolute quantication of these biomarkers in a single sample is always required for accurate diagnostics. [9][10][11][12][13][14] However, the absolute intensity of biomarkers obtained from the total probe concentration can be affected by physical factors such as poor washouts or uneven bindings, possibly giving rise to inaccurate readouts. 15,16 Moreover, specic standards are still required since the stoichiometry of the total probe concentration is hard to precisely determine.…”