Related Experiment Video
Updated: Sep 28, 2025
11:12
Identification of protein complexes with quantitative proteomics in S. cerevisiae
Published on: March 4, 2009
13.3K
A systematic evaluation of yeast sample preparation protocols for spectral identifications, proteome coverage and
Maxime den Ridder1, Ewout Knibbe1, Wiebeke van den Brandeler1
1Delft University of Technology, Department of Biotechnology, van der Maasweg 9, 2629 HZ Delft, the Netherlands.
Journal of Proteomics
|March 30, 2022
Summary
Optimizing sample preparation for yeast proteomics is crucial for accurate results. This study systematically compared protocols, finding moderate temperatures and times minimize artificial peptide modifications for better proteome analysis.
Area of Science:
- Proteomics
- Cellular Biology
- Biochemistry
Background:
- Accurate cellular proteomics is vital for understanding biological processes, especially in model organisms like yeast (Saccharomyces cerevisiae).
- Optimized sample preparation is critical for unicellular organisms with rigid cell walls, as existing protocols can introduce bias or artificial modifications.
- Yeast is a key model organism and cell factory, making its proteome dynamics under various conditions an important research area.
Purpose of the Study:
- To systematically investigate and compare various sample preparation protocols for whole-cell lysate, bottom-up proteomics in yeast.
- To evaluate the impact of different conditions on spectral identification, proteome coverage, and peptide modifications.
- To provide optimized protocols and raw data as a resource for future yeast proteomics studies.
Main Methods:
- A matrix of different sample preparation conditions was applied to Saccharomyces cerevisiae.
- Proteomics experiments utilized database and unrestricted modification search approaches.
- Evaluation metrics included identified spectra fraction, proteome and amino acid sequence coverage, GO-term distribution, and peptide modifications.
Main Results:
- Optimized protocols identified approximately 65-70% of acquired fragmentation spectra.
- De novo sequencing indicated unidentified spectra were of low quality.
- Moderate incubation temperatures and times were found to circumvent excessive formation of modification artifacts.
- Solvents, additives, and filter materials were linked to various peptide modifications.
Conclusions:
- The study provides a systematic evaluation of sample preparation protocols for yeast proteomics.
- Selected protocols and raw data serve as a valuable resource for designing new protocols and analyzing peptide modifications.
- Minimizing artificial modifications through optimized conditions is key to accurate proteome analysis in yeast.
Keywords:
Open modification searchProtein modificationsProteome coverageSample preparation protocolsYeastde novo sequencing
