High resolution profiling of cell cycle-dependent protein and phosphorylation abundance changes in non-transformed cells

The cell cycle governs a precise series of molecular events, regulated by coordinated changes in protein and phosphorylation abundance, that culminates in the generation of two daughter cells. Here, we present a proteomic and phosphoproteomic analysis of the human cell cycle in hTERT-RPE-1 cells using deep quantitative mass spectrometry by isobaric labelling. Through analysing non-transformed cells, and improving the temporal resolution and coverage of key cell cycle regulators, we present a dataset of cell cycle-dependent protein and phosphorylation site oscillation that offers a foundational reference for investigating cell cycle regulation. These data reveal uncharacterised regulatory intricacies including proteins and phosphorylation sites exhibiting previously unreported cell cycle-dependent oscillation, and novel proteins targeted for degradation during mitotic exit. Integrated with complementary resources, our data link cycle-dependent abundance dynamics to functional changes and are accessible through the Cell Cycle database (CCdb, https://slim.icr.ac.uk/cell_cycle/cell_cycle), an interactive web-based resource for the cell cycle community. 

Reference:

BioRxiv paper - High resolution profiling of cell cycle-dependent protein and phosphorylation abundance changes in non-transformed cells

Table description:

Table S1: Detailed information supporting Figure 1. Quantification of cells in each cell cycle phase based on DNA content by flow cytometry with propidium iodide staining. Analysis was performed on aliquots of samples used for mass spectrometry analysis. List of proteins from the UniProt Resource annotated with proteins detected in this study and other cell cycle proteomics datasets. Cyclins and proteins associated with the mitotic cell cycle process Gene Ontology (GO) term (GO:1903047) in publicly available datasets are reported.

Table S2: List of proteins and phosphorylation events detected in the Time Course dataset. Protein and phosphorylation abundance values are grouped by normalisation method. Statistical metrics and other functional annotations are reported. Phosphorylation sites are annotated with structural, evolutionary, functional, genomic and proteomics data. 

Table S3: Gene Ontology (GO) term ( “cell cycle” GO:0007049) enrichment analysis on different fold change ranges used to define the cutoffs for Cell Cycle-dependent (CCD) proteins in the Time Course and Mitotic Exit datasets.

Table S4: Gene Ontology (GO) term enrichment analysis on proteins and phosphorylation events significantly oscillating in the Time Course dataset categorised based on Biological Process, Molecular Function, Cellular Compartment and Reactome Pathway.

Table S5: List of proteins and phosphorylation events detected in the Mitotic Exit dataset. Protein and phosphorylation abundance values are grouped by normalisation method. Statistical metrics and other functional annotations are reported. Phosphorylation sites are annotated with structural, evolutionary, functional, genomic and proteomics data.

Table S6: Gene Ontology (GO) term enrichment analysis on proteins and phosphorylation events significantly differentially expressed in the Mitotic Exit dataset categorised based on Biological Process, Molecular Function, Cellular Compartment and Reactome Pathway.

Table S7: List of proteins found in clusters shown in Figure 3F along with Gene Ontology (GO) term enrichment analysis of these proteins categorised based on Biological Process, Molecular Function, Cellular Compartment and Reactome Pathway.

Table S8: Set of Cell Cycle-dependent (CCD) proteins and phosphorylation sites. Protein and phosphorylation abundance values are grouped by normalisation method. Statistical metrics and other functional annotations are reported. Phosphorylation sites are annotated with structural, evolutionary, functional, genomic and proteomics data. Other studies defining CCD proteins and phosphorylation sites are indicated. Proteins detected in other cell cycle cell cycle datasets together with CCD proteins defined in other studies.

Table S9: Gene Ontology (GO) term enrichment analysis of Cell Cycle-dependent (CCD) proteins and phosphorylation events categorised based on Biological Process, Molecular Function, Cellular Compartment and Reactome Pathway.

Table S10: Localisation and Protein Complex enrichment analysis shown in Figure 4B. The localisation analysis table contains the localisation GO terms, the number of the proteins assigned with the respective term along with the curve fold change mean, median and standard deviation calculated from the curve fold changes of the individual proteins for a particular localisation GO term. The Protein Complexes table contains the proteins involved in the complexes along with the curve fold change mean, median and standard deviation calculated from the curve fold changes of the individual proteins involved in the complex. 

Table S11: Proteins and transcripts identified in the Mahdessian et al annotated with Cell Cycle-dependent (CCD) proteins detected in this study.

Table S12: Phosphorylation sites localised at protein interfaces. Statistical metrics and other functional annotations are reported.

Table S13: Cycle-dependent (CCD) proteins known and predicted to contain degradation motifs. Prediction confidence values and proteins containing unstable peptides are reported.