Crucial insights into the fundamentals of single-cell biology
This study from the Olsen group at University of Copenhagen, has combined recent developments in both high-sensitivity instrumentation and sample preparation methods to perform single-cell pSILAC experiments. The authors have used this elegant approach to uncover new insights of protein function in relation to protein turnover at the single-cell level.
Proteins are the molecular machinery of living organisms that control and regulate all biological functions in a cell through signaling pathways and post-translational modifications. Protein turnover is defined by the balance between synthesis and degradation, and is an important mechanism to regulate biological processes and diseases, including cancer.
LC-MS based proteomics is the only technology capable of providing unbiased, and systems-wide measurements of protein synthesis, degradation, and turnover rates, through pulsed stable isotope labeling by amino acids in cell culture (pSILAC). However, pSILAC has so far been limited to bulk analysis of cell lines or tissues and can only estimate average protein turnover rates within a cell population, which frequently includes different cell types and states.
Deeper understanding of dynamic cellular processes in individual cells with pSILAC
Using state-of-the-art technology, the team demonstrated that two SILAC labels are detectable from ~4,000 proteins in single HeLa cells, recapitulating known biology.
Furthermore, they performed a large-scale time-series SC-pSILAC analysis of undirected differentiation of human induced pluripotent stem cells (iPSCs) encompassing 6 sampling times over 2 months and analyzed >1,000 individual single cells.
By combining pSILAC with the Chip-Tip based single-cell LC-MS workflows involving single cell dispensing and sample preparation using the cellenONE with a proteoCHIP EVO 96 and direct transfer to Evotips, and subsequent analysis using the Evosep One LC with Whisper flow gradients coupled to narrow-window DIA (nDIA) on the Orbitrap Astral mass spectrometer, they uncovered critical insights into protein turnover dynamics, identifying distinct regulatory mechanisms governing dividing and non-dividing cells.
This method enabled the detection of subtle differences in protein stability, an essential factor in cellular processes such as signal transduction, receptor recycling, and post-translational modifications. The ability to monitor these dynamics in single cells offers an unprecedented view into the heterogeneity of biological systems, highlighting key protein interactions that would otherwise remain obscured in bulk analyses.
First author Pierre Sabatier highlights:
”Our study introduces 2-dimensional measurements to single-cell proteomics. This work, particularly the large-scale analysis of stem cell differentiation, is a testament to the incredible developments currently happening in the field. The exciting part is that we can already envision much larger studies where multidimensional single-cell proteomics analysis will contribute to new insights into the regulation of protein and cellular functions.”
A leap forward for cellular and biomedical research
The study’s findings have significant implications for regenerative medicine, cancer research, and other fields where cellular heterogeneity plays a pivotal role. By offering a multidimensional perspective of protein regulation, this approach paves the way for deeper functional insights into disease mechanisms and therapeutic targets
The study was published in the leading journal, Cell and the last author and leading expert in proteomics, Jesper V. Olsen, highlights the significance of this advancement:
“For the first time, researchers can systematically capture both protein abundance and turnover at the single-cell level, providing true biological insight. The robustness of this technology enables high-throughput analysis, ensuring reliable and reproducible data for the community. This represents a major step forward for the field.”
With the rapid evolution of SCP technologies, the integration of approaches like pSILAC is set to revolutionize our understanding of cellular biology, ultimately bringing us closer to personalized medicine and targeted therapies.
Meet users in biomedical research
Across biopharma, proteomics is helping to accelerate drug discovery, improve therapeutic targeting, and advance immunopeptidomics research. Many labs are using LC-MS to better understand disease mechanisms and develop new treatments. Join our upcoming webinars to learn more about how LC-MS is being used in biopharma research here
Read full publication in Cell here or visit our Single Cell application page to learn more
