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Single-cell proteomics (SCP) has emerged as a transformative approach for directly quantifying protein expression at the level of individual cells, addressing a key knowledge gap in traditional bulk proteomics. _While bulk methods average protein profiles across large cell populations, they obscure the cellular heterogeneity and rare cell populations that drive biological processes such as development, differentiation, disease progression, and treatment respons_e. However, despite methodological advances in recent years, practical best practices for end-to-end SCP workflows remain under active optimisation, particularly regarding the integration of sample preparation, liquid chromatography, and mass spectrometry parameters.
Suárez-Fernández et al. optimised a complete label-free single-cell proteomics protocol to address these workflow integration gaps. The team systematically evaluated the complete workflow, from cell dispensing using the Tecan UNO thermal-inkjet platform through LC separation on a Vanquish Neo system coupled to an Orbitrap Exploris 480 mass spectrometer with FAIMS Pro interface. Two data-independent acquisition (DIA) schemes were compared: MS2-DIA and HRMS1-DIA.
This quantitative study demonstrated significant improvements in protein identifications across various conditions, with the optimised workflow achieving up to 3,500 protein groups from single human mesenchymal stem cells (hMSCs). The Aurora® Elite™ 15×75 C18 UHPLC column at 40 samples per day outperformed the PepMap Neo column, yielding over 3,000 protein groups per cell. Pathway and gene ontology analyses confirmed broad coverage of human biological pathways and subcellular compartments, including low-abundance organelles.
The findings highlight that tailoring LC and MS parameters to specific experimental needs, rather than applying one-size-fits-all approaches, is essential for maximising proteome depth and functional coverage in single-cell analyses. This accessible workflow facilitates adoption across general proteomics laboratories and core facilities.
Publication
bioRxiv
Authors
Amanda Suárez-Fernández, Judit Bestilleiro-Márquez, Gonzalo Sánchez-Duffhues, & Ignacio Ortea;
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