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

The analysis of ultra-low input samples or individual cells is critical for answering a multitude of biomedical questions. But current proteomic workflows are limited in their sensitivity and reproducibility. Here we report a complete workflow that includes optimized strategies for cell lysis to data analysis. Thanks to the use of a convenient to handle volume of 1 µL and standardized 384 well plates, the workflow is easy to implement, even for newcomers to the field. At the same time, the workflow can be performed in a semi-automated fashion using the CellenONE® robot, which allows for high reproducibility. Ultrashort gradient lengths ranging to little as 5 min for highest possible throughput were tested with advanced µ-pillar columns. DDA, WWA and DIA as well as commonly used and advanced data analysis algorithms were benchmarked. From a single cell, 1790 proteins were identified using DDA and a dynamic range of 4 orders of magnitude was covered. Using DIA, the proteome coverage was increased to >2200 proteins from single cell level input within a 20 min active gradient. We applied our workflow to differentiate two cell lines showing its ability to investigate cellular heterogeneity as proof of principle. ### Competing Interest Statement The authors have declared no competing interest.