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Shotgun Proteomics Sample Processing Automated by an Open-Source Lab Robot
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Automating Complex, Multistep Processes on a Single Robotic Platform to Generate Reproducible Phosphoproteomic Data.
B Todd Mullis1, Sunil Hwang2, L Andrew Lee3
1Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC, USA.
SLAS Discovery : Advancing Life Sciences R & D
|September 27, 2019
Summary
Automating sample preparation for mass spectrometry-based phosphoproteomics is crucial for clinical use. This study presents a fully automated method for peptide desalting and phosphopeptide enrichment, improving identification and reproducibility.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Proteomics
Background:
- Mass spectrometry-based phosphoproteomics is vital for disease diagnosis and drug development.
- Clinical translation is hindered by unstandardized, segmented sample preparation.
- Automation of the entire process is essential for standardization.
Purpose of the Study:
- To develop a fully automated method for peptide desalting and phosphopeptide enrichment.
- To integrate sample preparation steps for improved efficiency and standardization.
- To facilitate the clinical translation of phosphoproteomics.
Main Methods:
- Utilized IMCStips on a Hamilton STAR robotic platform for automated extraction.
- Developed an integrated workflow for peptide desalting and phosphopeptide enrichment.
- Assessed method reproducibility using multiple reaction monitoring (MRM).
Main Results:
- Identified over 10,000 phosphopeptides from 200 µg HCT116 cell lysate with >85% specificity.
- Achieved 50% higher phosphopeptide identifications compared to titania-based methods.
- Demonstrated >50% phosphopeptide recovery with <20% CVs over 3 weeks.
Conclusions:
- The automated IMCStips method offers a significant advancement in phosphoproteomics sample preparation.
- This automation contributes to the standardization of phosphopeptide analysis.
- The method provides a foundation for a fully automated 'cells to phosphopeptides' workflow.
