The lab is very excited to introduce Single Cell Proteomics analysis as a service starting in July 2025!
We utilize the CellenOne liquid handler and the nPOP (nanodroplet processing in one pot) technology (link), optimized in collaboration with Scienion. Data are acquired in our Orbitrap Exploris 480 with FAIMS module coupled to nano liquid chromatography. The entire system is dedicated to single cell experiments. The acquisition method is pSCOPE (link), which utilizes MaxQuant.Live (link) to prioritize MS/MS events to minimize missing values. For histone analysis, the experiment is performed on the timsTOF HT in a label-free mode.
With single cell proteomics data, it is already possible to (i) define different cell types and cell states beyond the current single markers used today for e.g. FACS sorting; (ii) perform pseudotime inference analyses to gain insight on continuous processes such a cell differentiation; (iii) quantify protein co-variation to gain insight on regulatory mechanisms; (iv) integrate protein and RNA measurements from the same biological systems to predict transcriptional and post-transcriptional regulation. An excellent overview of the applications of this analysis is published here (link).
Technical details for the experiment
The Sidoli lab only needs a suspension of single cells (fresh or frozen) after gentle fixation to “block” biological events. The lab then performs single cell preparation with the CellenOne, TMT labeling using the 32plex, generates a carrier to boost sensitivity and performs LC-MS/MS followed by data analysis using R packages.
Samples are recommended in groups of approx. 1,000 cells, with a maximum of 4,000 cells per batch. For instance, we can test 10 conditions (100 cells each) at affordable prices. Or, we can study 4 conditions (1,000 cells each) for in depth assessment of population heterogeneity.
In regular experiments, we quantify approx. 1,000 proteins per cell, but improvements are occurring constantly.
Single cell histone modificationS
We also offer single cell histone analysis experiments to map the heterogeneity of post-translational modifications cell by cell. In our recent manuscript (link), we demonstrate that this technology can be used to identify cross-talk between histone modifications, predict which chromatin markers define regions or more/less stable chromatin, and cluster cells based on their differential response to drug treatment.
Special Acknowledgments
The development of this service would have not been possible without the critical help of multiple lab members and externals. A special thank you goes to:
Our team: Jennifer Aguilan for experimental planning and costs; Ronald Cutler for unconventional experimental design; Giulia Barotti for questions about throughput and sample multiplexing; Maxwell Horton for specific inquiries about preparing single cells from solid tissues; Carlos Madrid-Aliste for bioinformatics.
The Genetics team: Jan Vijg for providing access to the CellenOne; Bernice Morrow for external support; Jidong Shan for training on the instrument.
Scienion: Joshua Cantlon and Sonu Kumar for unconditional constant support for sample preparation.
Others: Andrew Leduc for coming to our lab and train us on nPOP, and being constantly available for consultation; Nikolai Slavov for being an invaluable member of Ronald Cutler and all of us.