Standardizing single cell proteomics with the Chip-Tip workflow
Proteins are the most dynamic molecules in the body, directly influencing cellular health and function. They are also the primary targets for most therapeutic treatments. Through proteomics, we can gain valuable insights into drug efficacy and safety by monitoring proteins in real-time. Significant progress has been made in this field over the past few decades, with recent technological advancements – particularly in the last five years – dramatically increasing both the speed and robustness of proteomics. These breakthroughs have turned proteomics into a powerful tool, and enhanced sensitivity now allows for the realization of single-cell proteomics (SCP), a frontier that promises to revoluionize our understanding of cellular diversity. SCP could significantly impact drug development and offer new opportunities for repurposing drugs in personalized treatment plans – unlocking a previously unexplored layer of protein-level information.
SCP is an emerging and rapidly advancing research field, but it faces inherent challenges. Unlike RNA, proteins cannot be amplified, making the small sample size particularly difficult to analyze. Each sample must undergo complex processes of lysis and digestion before being subjected to LC-MS analysis. Every step can potentially be associated with sample losses, but researchers have made significant breakthrough by fine-tuning every step from initial preparation all the way to mass spectrometry analysis.
Reaching an inflection point in SCP – moving beyond golden runs to gaining biological insight
A collective effort from the technological community has led to the development of several SCP workflows with impressive identification rates. However, these workflows are often highly specialized, requiring expert-level knowledge for successful implementation. We have now reached a key milestone, where the technology can deliver important biological insights, but standardization and higher throughput are needed to make SCP widely applicable.
To address these challenges, researchers from the Olsen lab at the University of Copenhagen have introduced the Chip-Tip workflow, a nearly loss-less SCP method capable of identifying over 5,000 proteins and 40,000 peptides from single HeLa cells. Their workflow involves single cell dispensing and sample preparation using the cellenONE platform, combined with the proteoCHIP EVO 96. The sample is then transferred directly to an Evotip and analyzed using the Evosep One with Whisper Zoom methods, employing narrow window DIA (nDIA) on the Orbitrap Astral mass spectrometer.
The proteoCHIP EVO 96 allows for the simultaneous processing of up to 96 samples in parallel and the design ensures optimal success in dispensing individual cells into separate wells. Furthermore, the proteoCHIP is tailored for seamless integration with the Evotip eliminating the need for additional pipetting steps and ensuring high efficiency and precision throughout the workflow. The Evotip serves as the sample introduction device to the Evosep One, capturing and storing the sample on C18 resin. This method prevents sample loss – common in traditional LC systems, especially when scaling up – ensuring maximum yield and accuracy.
This workflow leverages the robust Whisper Zoom methods and the sensitivity of the Orbitrap Astral mass spectrometer, resulting in a commercially available, standardized end-to-end solution that prioritizes both robustness and ease-of-use. This will enable more labs to implement SCP without requiring highly specialized expertise or extensive workflow optimization. This shift towards standardized, scalable solutions opens new opportunities for researchers to address critical biological questions more efficiently.
Unleashing the biological potential of the Chip-Tip workflow – analyzing non-directed human-induced pluripotent stem cell differentiation
The enhanced sensitivity and throughput of the Chip-Tip workflow offers wide-ranging applications in both basic biology and biomedicine. In this study, the authors demonstrate its use in analyzing the differentiation of human-induced pluripotent stem cells (hiPSCs) into various cell types. This analysis uncovered key stem cell markers and lineage-specific proteins, providing valuable insights into cellular differentiation and development.
The workflow’s robustness and scalability make it suitable for high-throughput studies, enabling the processing of up to 120 label-free SCP samples per day. This capacity is essential for large-scale studies, that aim to uncover the complexities of cellular functions and disease mechanisms.
In conclusion, the Chip-Tip workflow sets a new benchmark in SCP for its robustness, sensitivity, and throughput. The shift from striving for golden runs to delivering standardized biological insights marks a significant turning point in SCP. As technologies like the Chip-Tip workflow continue to evolve, the proteomics community is well-positioned to make even greater strides in understanding the complexities of biological systems.
What’s next?
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Read the full story in Nature: https://www.nature.com/articles/s41592-024-02558-2