Description
Cell cycle progression is controlled by temporally regulated changes in protein properties, including their abundance and post-translational modification. Mass spectrometry has played a pivotal role in revealing these changes in a comprehensive manner, mostly in the context of model organisms or human cancer cell lines that have been synchronised using small molecule inhibitors.Recently we demonstrated a novel method for cell cycle regulated proteome analysis that 1) avoids synchronization, 2) is compatible with primary cells, and 3) can isolate rare, transient phases of the cell cycle with biochemical specificity in high purities (Ly et al., eLife 2017). Human cells immunostained for intracellular cell cycle markers (cyclin A, cyclin B and H3S28ph) are sorted by FACS into 8 interphase and 8 mitotic populations. The interphase populations differ in cyclin A and cyclin B levels. Detailed separation of mitosis into prophase, early prometaphase, late prometaphase, anaphase, and telophase populations is possible by sorting by CycA-/+, CycB-/+, levels of H3S28ph and cell width. These populations are purified from an asynchronous culture of human TK6 cells, which are karyotype-stable B-cell lymphoblastoid cells. We call this method PRIMMUS, or proteomics of intracellular immunostained subsets.
Proteome wide analysis is technically challenging for the rarest populations. We therefore optimised the sample processing workflow and developed state of the art MS acquisition to measure >6,000 proteins from 2,000 cells. We then applied PRIMMUS to the sixteen cell cycle populations described above. We identify known cell cycle regulated proteins, including ribonucleotide reductase 2 (RRM2). We also uncover novel cell cycle regulated proteins that show decreased abundance at specific mitotic subphases. In future, we aim to investigate the developmental and tissue-specific plasticity in cell cycle regulation by applying PRIMMUS to compare the cell cycle regulated protein network in different primary cell types.
| Period | 20 Nov 2019 |
|---|---|
| Visiting from | University of Edinburgh (United Kingdom) |
| Visitor degree | PhD |
| Degree of Recognition | Regional |