Leveraging TMT multiplexing for increased throughput in protein-centric characterizations of heterogeneity at the single cell level

In recent years, life science research has experienced a significant shift, moving away from conducting bulk cell interrogation towards single-cell analysis. It is only through single-cell analysis that a complete understanding of cellular heterogeneity, and the interplay between various cell types fundamental to specific biological phenotypes can be achieved. The blood system is a prime example of such a complex hierarchy, where Hematopoietic stem cells are the origin of all cell lineages contained therein. Acute myeloid leukemia (AML), a perturbed state of hematopoiesis, is also hierarchically organized, with leukemia stem cells at the apex. Successful eradication of AML will likely depend on specific targeting of these tumour-initiating cells, in turn requiring their molecular characterization.

We have spent the past few years optimizing a proteomics workflow for analyzing single cells using Mass Spectrometry (scMS). Through isobaric labeling using latest-generation TMTPro reagents, we ensure highest possible cell throughput, bringing in-depth characterizations of >10,000 single cells within reach. We demonstrate both through pre-enrichment of cell populations and through a non-enriched unbiased approach that our workflow enables the exploration of cellular heterogeneity within healthy and aberrant blood hierarchies. We leverage a combination of fluorescence-activated cell sorting, automated liquid handling, next-generation chromatography, state-of-the-art MS instrumentation and complex data analysis workflows to provide a comprehensive suite of scMS tools. Combined, our workflow strikes a careful balance between cell throughput, proteome depth, quantitative accuracy and ease-of-implementation, and demonstrates the power of implementing global single-cell proteomics studies in labs across the world.