Poster Presentation 31st Annual Lorne Proteomics Symposium 2026

An End-to-End Workflow for Sensitive Single Cell Proteomics at 95 SPD (#127)

Nicolas Hartel 1 , Mikayla Shanafelt 1 , Dylan Multari 2 , Chris Xu 2 , Michelle Cielesh 3 , Angela Connolly 3 , Ben Crossett 3 , Stuart Cordwell 3 , Ralf Schittenhelm 2 , Daniel Hermanson 1 , Mark Larance 3
  1. Thermo Scientific, San Jose, USA
  2. Monash University, Clayton, Vic
  3. The University of Sydney, Camperdown, NSW, Australia

Single-cell proteomics (SCP) is a rapidly advancing field that enables quantification of protein abundance with high accuracy and provides enhanced resolution of cellular heterogeneity compared to bulk cell or tissue analyses. This study evaluates the performance of the Orbitrap Astral Zoom mass spectrometer for SCP, integrated with FAIMS Pro Duo interface ion pre-filtering and high-throughput liquid chromatography using the Vanquish Neo UHPLC system at speeds up to 95 samples per day. Isolated HeLa cells were digested with trypsin and processed using a streamlined protocol directly in 384-well plates. Employing a data-independent acquisition (DIA) strategy, we reproducibly identified approximately 4,000 proteins from ~22,000 peptides in single HeLa cells. MS1-based quantification revealed a dynamic range exceeding four orders of magnitude in protein abundance, with less than 20% missing values across 164 single-cell datasets. Analysis with and without cross-run normalization revealed protein abundance variations associated with cell size and G2/M-phase markers. Global correlation analysis identified enrichment of highly co-expressed protein complexes, including the MCM complex, ribosome, proteasome, ATP synthase, and mitochondrial electron transport components. These findings demonstrate a simplified and scalable SCP workflow, highlighting the sensitivity and reproducibility of the Orbitrap Astral Zoom MS platform for high-throughput single-cell proteomic analysis.