Robust and easy-to-use one pot workflow for label free single cell proteomics

The analysis of ultra-low input samples or even individual cells is essential to answering a multitude of biomedical questions, but current proteomic workflows are limited in their sensitivity and reproducibility. Here we report a comprehensive workflow that includes optimized strategies for all steps from cell lysis to data analysis. Thanks to convenient to handle 1 L sample volume and standardized 384 well plates the workflow is easy for even novice users to implement. At the same time, it can be performed semi-automatized using the CellenONE(R), which allows for highest reproducibility. To achieve high throughput, ultrashort gradient lengths down to 5 min were tested using advanced -pillar columns. Data-dependent acquisition (DDA), wide-window acquisition (WWA) and data-independent acquisition (DIA), and commonly used advanced data-analysis algorithms were benchmarked. Using DDA, 1790 proteins covering a dynamic range of four orders of magnitude were identified in a single cell. Using DIA, proteome coverage increased to more than 2200 proteins identified from single cell level input in a 20-min active gradient. The workflow enabled differentiation of two cell lines, demonstrating its suitability to cellular heterogeneity determination.