Single-cell proteomics (SCP) has gained significant attention in recent years. This technique allows for the differentiation of proteomic profiles at the single-cell level, which is not possible with traditional bulk proteomics, where the information of each protein is averaged across all cells in the sample. This enables the exploration of diversity and state among subpopulations of cells with distinct expression profiles. The potential applications of SCP are highly diverse, including cancer research, where it can characterize the mechanisms involving different cell types; stem cell biology, to understand the processes of pluripotency and differentiation into various cell types; and precision medicine, to understand individual patient variability in response to treatments.

Given the extremely low amount of protein contained in a single cell, the two critical points in SCP are sample preparation and LC-MS analysis. Only recently have mass spectrometers been developed with sufficient sensitivity to achieve acceptable cellular proteome coverage. Consequently, SCP is a very recent field, and most studies conducted to date focus on the development and optimization of methods for individual cell dispensing, processing (cell lysis and protein digestion), and LC-MS analysis. The use of automated platforms increases robustness and decreases variability in the results, in addition to promoting the standardization of protocols.

In this study, we used a relatively affordable automated platform, the Tecan UNO (HP D100), for the dispensing of single cells and their subsequent processing in a single step (cell lysis and rapid enzymatic digestion). Human mesenchymal cell digests were prepared in 96-well plates, with a total processing time of less than 75 minutes (including enzymatic digestion with Trypsin and Lys-C). The plates were loaded directly, or after freezing, into the autosampler of a Vanquish Neo LC system (Thermo Scientific), avoiding sample transfers. Various commercial chromatographic columns were tested in standard setups as used for bulk proteomics, with a throughput of approximately 48 samples per day. MS analysis was performed in an Orbitrap Exploris 480 (Thermo Scientific), with a nanoFlex source and FAIMS Pro Duo ion mobility interface. Several DIA acquisition methods, optimized for low-amount protein samples and using one FAIMS compensation voltage, were tested, and the runs were processed with Spectronaut v17 in the directDIA (library-free) workflow. For functional analysis and assessment of the molecular pathways covered by the identified proteins, STRING v12.0 and Reactome v88 were used to obtain pathway and process coverage for the identified protein groups.

The results obtained regarding the single cell dispensing success, identification and quantification of proteins with the different variables tested will be presented, along with the GO terms and molecular pathways covered by the optimized workflow.