Single-cell proteomics (SCP) holds enormous promise for resolving cellular heterogeneity at the protein level, yet the field remains limited by low sensitivity, incomplete proteome coverage, and high rates of missing data – particularly for low-abundance proteins. These gaps are especially problematic when studying subtle biological differences, such as drug-induced responses or cell maturation states. Janssens et al. aimed to evaluate whether a newly engineered ion processing system, the Athena Ion Processor (AIP) integrated into the timsTOF Ultra 2 framework (timsUltra AIP), could address these limitations in a biologically relevant single-cell context.

The researchers performed global, untargeted DIA proteomics (dia-PASEF) on human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) across a controlled cell dilution series, benchmarking the timsUltra AIP against the standard timsTOF Ultra 2. Peptides were separated on an Aurora® Elite™ 15×75 CSI C18 UHPLC column coupled to a nanoElute 2. At the single-cell level, the timsUltra AIP delivered more than a two-fold increase in protein identifications (409 vs. 1,039 proteins per cell), reduced data missingness, and improved quantitative reproducibility, with over 75% of proteins quantified below 20% coefficient of variation. Approximately 50% of the total detected proteome was exclusively identified using the timsUltra AIP.

Leveraging this enhanced workflow, the researchers demonstrated that iPSC-CMs exist as at least two proteomic subtypes: mature cardiomyocytes and immature cells. They also demonstrated that these subtypes respond differently to the Parkin activator PR-364, with mature cells showing dose-dependent enrichment of mitochondrial and metabolic pathways, while immature cells exhibited more variable, structurally-focused responses. These findings highlight how deeper single-cell proteome coverage can uncover drug response heterogeneity that would otherwise be obscured in bulk analyses, with direct implications for pre-clinical cardiac drug development.

Publication
bioRxiv

Authors

Johannes V Janssens, Aleksandra Binek, Lizhuo Ai, Ajay Bharadwaj, Madelyn Arzt, Diego Assis, Matthew Willetts, Michael Krawitzky, Daniel Hornburg, Arun Sharma, Aleksandr Stotland, & Jennifer E. Van Eyk;

Title

Expanded proteome coverage powered by advanced ion processing enables deep single-cell drug response subtyping in human stem cell derived cardiomyocytes