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bioRxiv · 10.1101/2025.10.01.679734

Accessible and fast single-cell proteomics by DIA integrating Tecan UNO cell dispensing platform, Vanquish Neo LC and Exploris 480 MS

Suarez-Fernandez, A.·Bestilleiro-Marquez, J.·Sanchez-Duffhues, G.·Ortea, I.
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Abstract

Recent technological advancements in high-sensitivity LC-MS platforms and MS acquisition strategies have addressed key analytical challenges in single-cell proteomics (SCP), paving the way for SCP to become a transformative tool in biology and medicine. Techniques such as MS2 DIA and HRMS1 DIA exploit wide isolation windows and high-resolution MS1 and MS2 spectra to maximize protein identifications, even from low-abundance signals. In parallel, improved data processing algorithms reduce missing values and enhance the reliability of SCP data. These advancements are complemented by automated cell isolation technologies, which ensure precise single-cell handling with minimal sample loss, and in some implementations enable integrated, automated downstream sample preparation steps. Despite this progress, practical, end-to-end best practices for MS-based SCP remain an active area of optimization. Severe sample limitation, adsorption-related losses, the need for robust quantification across hundreds to thousands of single cells, and the lack of comprehensive protocols that systematically integrate and compare critical workflow variables complicate method selection and SCP implementation. In this context, this study builds on recent LC-MS based SCP advancements by presenting a complete, optimized, minimum user operation SCP workflow that integrates the Tecan UNO cell dispensing platform, a Vanquish Neo LC system, and an Orbitrap Exploris 480 mass spectrometer, applied to human primary cells. The workflow is designed to minimize data/cell loss, ensure high reproducibility, and maximize identifications. We evaluate key parameters, including acquisition schemes and LC gradient and column, and distill practical recommendations. The resulting protocol provides a foundation for an easy and accessible routine SCP application in biology and medicine.

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Suarez-Fernandez, A., Bestilleiro-Marquez, J., Sanchez-Duffhues, G., Ortea, I.. 2025-10-02. Accessible and fast single-cell proteomics by DIA integrating Tecan UNO cell dispensing platform, Vanquish Neo LC and Exploris 480 MS. https://doi.org/10.1101/2025.10.01.679734

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