Workflow and results of Chip-Tip SCP. From Ye et al., 2025. “Enhanced sensitivity and scalability with a Chip-Tip workflow enables deep single-cell proteomics“, Nat Methods 22, 499–509 (2025). https://doi.org/10.1038/s41592-024-02558-2. Licensed under the terms of the Creative Commons CC-BY 4.0 license.

Existing single-cell proteomics techniques are limited by low protein identification numbers, low sensitivity, and challenges in sample preparation. Ye et al. developed the Chip-Tip method to address these knowledge gaps, aiming to significantly improve protein identification and characterisation from single cells.

Their experimental approach involved developing an ultra-sensitive workflow combining innovative sample preparation (using cellenONE X1 platform and proteoCHIP EVO 96), advanced liquid chromatography (Evosep One LC with Whisper flow gradients), and mass spectrometry (Orbitrap Astral with narrow-window data-independent acquisition).

The method used Aurora Elite XT 15×75 C18 UHPLC column with an EASY-Spray ion source, improving protein identification from approximately 2,000 to 5,000-6,500 proteins per single cell. By enabling deep protein coverage without specific enrichment, the researchers could analyse post-translational modifications like phosphorylation and glycosylation directly. The technique was demonstrated through two biological applications: analysing cancer cell spheroids after 5-fluorouracil treatment and tracking human-induced pluripotent stem cell differentiation.

This breakthrough represents a significant leap in single-cell proteomics, potentially transforming our understanding of cellular heterogeneity, disease mechanisms, and developmental processes.

Publication
Nature Methods

Authors

Zilu Ye, Pierre Sabatier, Leander van der Hoeven, Maico Y. Lechner, Teeradon Phlairaharn, Ulises H. Guzman, Zhen Liu, Haoran Huang, Min Huang, Xiangjun Li David Hartlmayr, Fabiana Izaguirre Anjali Seth, Hiren J. Joshi Sergey Rodin, Karl-Henrik Grinnemo, Ole B. Hørning, Dorte B. Bekker-Jensen, Nicolai Bache, & Jesper V. Olsen;

Title

Enhanced sensitivity and scalability with a Chip-Tip workflow enables deep single-cell proteomics