Evaluation of a transfer-free proteomics approach for the analysis of few dispensed cells: a core facility perspective

The field of single-cell proteomics has witnessed significant advancements in recent years. However, despite these advances, single-cell proteomics remains a challenging area for laboratories with limited resources, including personnel, instrumentation, and investments. This is due to the inherent difficulties associated with achieving optimal sample preparation, throughput, and sensitivity, which are essential for obtaining a comprehensive proteome profile. A recent study by Ye et al. [1] introduced a novel workflow for direct cell dispensing in an Evotip (Evosep) followed by on-tip lysis, protein digestion, and clean-up using an Uno Single Cell Dispenser^TM. This study aims to investigate its application in a core facility setting for samples ranging between 1 and 25 cells.

HeLa cells at near confluency were washed twice with DPBS and the cell suspension was filtered to remove debris. The cell viability and concentration were determined and diluted to a working concentration of 200 cells/μl. Twenty microliters of solution were transferred to a C1 Dispensehead Cassette (Tecan) and loaded in the Uno Single Cell Dispenser. The cells were then directly dispensed on a pre-equilibrated Evotip with an aqueous layer. Enzymatic digestion was performed on the Evotip and subsequently prepared for LC-MS analysis on an Evosep (EvoSep Biosystems) coupled either to an Orbitrap Astral (Thermo) or a timsTOF (Bruker). Both datasets will be analyzed using the directDIA approach in Spectronaut (Biognosys).

Evotips Pure were equilibrated by preparing a buffering layer on the upper part of the tip. Cells were dispensed directly into the Evotip using the Uno Single Cell Dispenser and a single-cell success dispensing rate >95% was achieved. For processing 96 samples, cells were dispensed in under 5 min resulting in an overall sample preparation time of 5h, including 4h enzymatic digestion. Once the settings are optimized, the workflow is universally applicable to cell types within diameters of 9‑25 μm. To benchmark the workflow, single cells were also dispensed with the Uno Single Cell Dispenser as well as FACS in 384-well plates, digested with the help of a Mosquito HV (SPT Labtech) and manually loaded into pre-conditioned Evotips. Data were acquired on the Orbitrap Astral mass spectrometer and will be acquired on a timsTOF in DiaPASEF mode both coupled to an Evosep One LC device. Preliminary results demonstrate that a higher level of reproducibility is achieved when dispensing single cells in an aqueous layer compared to an empty Evotip. However, the on-Evotip dispensing approach led to lower identifications than the in-plate methods. The preliminary results show that similar data are obtained from cells processed in-plate after dispensing with the Uno Single Cell Dispenser or the FACS sorter demonstrating that the Uno Single Cell Dispenser is well-suited for implementation in a core facility.

[1] Ye, Z., et al., Nat Commun 15, 2474 (2024)