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Simple In-Cell Processing Enables Deep Proteome Analysis of Low-Input Caenorhabditis elegans
Malek Elsayyid1, Jessica E Tanis1, Yanbao Yu2
1Department of Biological Sciences, University of Delaware, Newark, Delaware 19716, United States.
Analytical Chemistry
|April 21, 2025
Summary
We developed a new on-filter in-cell (OFIC) method for faster and more efficient protein extraction from Caenorhabditis elegans. This approach allows for comprehensive proteome analysis even with low-input samples, revealing insights into gene function.
Area of Science:
- Proteomics
- Model Organisms
- Molecular Biology
Background:
- Caenorhabditis elegans is a key genetic model organism.
- The worm cuticle hinders intracellular protein extraction for proteomics.
- Conventional methods are time-consuming and cause sample loss, especially for low-input samples.
Purpose of the Study:
- To develop a streamlined, efficient method for Caenorhabditis elegans proteomics.
- To overcome challenges associated with protein extraction from C. elegans.
- To enable low-input and single-worm proteome analysis.
Main Methods:
- Developed an on-filter in-cell (OFIC) processing approach for methanol-fixed C. elegans.
- Utilized single-shot liquid chromatography-mass spectrometry (LC-MS).
- Compared OFIC performance against conventional lysis-based methods and applied it to sod-1 mutant analysis.
Main Results:
- Identified over 9400 proteins from 200 worms using OFIC, the largest C. elegans proteome reported without fractionation.
- OFIC demonstrated superior performance in protein identification and quantitation compared to lysis methods.
- Loss of sod-1 impacts proteins involved in stress response, ribosome biogenesis, and metabolism.
Conclusions:
- The OFIC approach offers a simplified, low-sample-loss workflow for C. elegans proteomics.
- This method is effective for low-input samples, including single worms.
- The OFIC approach is broadly applicable to other biological systems.
