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 3500 yeast proteins from the in-cell digests. Our study introduces a novel in-cell approach for yeast proteomics analysis and presents a quantitative proteome map of yeast cell-cycle progression with high temporal resolution for cell division cycle (Cdc) proteins. It also provides a comprehensive, time-resolved view of proteome-wide dynamics and remodeling throughout the yeast cell cycle in response to methyl methanesulfonate (MMS)-induced DNA damage. SUMMARY: Yeast proteomics studies often require detergent-based and/or mechanical disruption procedures for cell lysis and protein digestion. We reported an "in-cell proteomics" approach that eliminates cell lysis and digests proteins directly in the yeast cells after a simple methanol fixation. The approach integrates all the sample processing into a single filter device, offering a rapid yet highly effective and sensitive approach for yeast proteomics analysis. Using this method, we were able to characterize proteome dynamics in the budding yeast Saccharomyces cerevisiae during cell cycle progression and following DNA damage.

• PMID: 41410420
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Journal Article

Authors

Nwaora H, Yu Y, Zhuang Z

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