Dataset Information

In-cell proteomics enables high-resolution temporal profiling of cell cycle progression and DNA damage response in Saccharomyces cerevisiae

ABSTRACT: Yeast is a widely used model organism in biological and proteomics research. Conventional bottom-up proteomic analysis of yeast cells requires disruption of the rigid cell wall to extract proteins, which is often associated with lengthy procedures, significant technical variations and noticeable sample loss. Here, we present an “in-cell proteomics” approach that eliminates cell lysis and digests proteins directly in the yeast cells after a rapid methanol fixation. The approach integrates all the sample processing into a single filter device, offering a simple yet highly effective and sensitive approach for yeast proteomics analysis. We applied this approach to characterize proteome dynamics in the budding yeast Saccharomyces cerevisiae during cell cycle progression and following DNA damage. With single-shot LC-MS, we were able to detect and quantify around 3,500 yeast proteins from the in-cell digests. Our study presents a quantitative proteome map of yeast cell-cycle progression with high temporal resolution for cell division cycle (Cdc) proteins and introduces a novel in-cell proteomics approach for yeast. It also provides a comprehensive, time-resolved view of proteome-wide dynamics and remodeling throughout the yeast cell cycle and in response to methyl methanesulfonate (MMS)-induced DNA damage.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Saccharomyces Cerevisiae (ncbitaxon:4932)

SUBMITTER: Zhihao Zhuang

PROVIDER: MSV000097997 | MassIVE | Mon May 26 20:04:00 BST 2025

REPOSITORIES: MassIVE

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In-Cell Proteomics Enables High-Resolution Temporal Profiling of Cell Cycle Progression and DNA Damage Response in Saccharomyces cerevisiae.

Nwaora Henry H Yu Yanbao Y Zhuang Zhihao Z

Proteomics 20251218

Yeast is a widely used model organism in biological and proteomics research. Conventional bottom-up proteomic analysis of yeast cells requires disruption of the rigid cell wall to extract proteins, which is often associated with lengthy procedures, significant technical variations, and noticeable sample loss. Here, we present an "in-cell proteomics" approach that eliminates cell lysis and digests proteins directly in the yeast cells after a rapid methanol fixation. The approach integrates all th ...[more]

PMID: 41410420

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Project description:In the budding yeast Saccharomyces cerevisiae, transcription factors (TFs) regulate the periodic expression of many genes during the cell cycle, including gene products required for progression through cell-cycle events. Experimental evidence coupled with quantitative models suggest that a network of interconnected TFs is capable of regulating periodic genes over the cell cycle. Importantly, these dynamical models were built on transcriptomics data and assumed that TF protein levels and activity are directly correlated with mRNA abundance. To ask whether TF transcripts match protein expression levels as cells progress through the cell cycle, we applied a multiplexed targeted mass spectrometry approach (parallel reaction monitoring) on synchronized populations of cells. We found that protein expression of many TFs and cell-cycle regulators closely followed their respective mRNA transcript dynamics in cycling wild-type cells. Discordant mRNA/protein expression dynamics were also observed for a subset of cell-cycle TFs and for proteins targeted for degradation by E3 ubiquitin ligase complexes such as SCF (Skp1/Cul1/F-box) and APC/C (anaphase-promoting complex/cyclosome). We further profiled mutant cells lacking B-type cyclin/CDK activity (clb1-6), where oscillations in ubiquitin ligase activity, cyclin/CDKs, and cell-cycle progression are halted. We found that a number of proteins were no longer periodically degraded in clb1-6 mutants compared to wild type, highlighting the importance of post-transcriptional regulation. Finally, the TF complexes responsible for activating G1/S transcription (SBF and MBF) were more constitutively expressed at the protein level than their periodic mRNA expression levels in both wild-type and mutant cells. This comprehensive investigation of cell-cycle regulators reveals that multiple layers of regulation (transcription, protein stability, and proteasome targeting) affect protein expression dynamics during the cell cycle.