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Inserting Pre-Analytical Chromatographic Priming Runs Significantly Improves Targeted Pathway Proteomics With Sample
Steven R Shuken1, Qing Yu1, Steven P Gygi1
1Department of Cell Biology, Harvard Medical School, 240 Longwood Ave, Boston, MA 02115, USA.
Biorxiv : the Preprint Server for Biology
|February 19, 2024
Summary
GoDigViewer software improves targeted proteomics by introducing "priming runs," significantly increasing peptide quantification success rates for complex protein assays.
Area of Science:
- Proteomics
- Mass Spectrometry
- Computational Biology
Background:
- GoDig is a targeted proteomics platform using tandem mass tags (TMT) for high throughput.
- Limited peptide-level quantification success hinders analysis of challenging proteins.
Approach:
- Developed GoDigViewer software for detailed visualization of GoDig runs.
- Implemented a new acquisition mode using "priming runs" for improved elution order calibration.
Key Points:
- Priming runs significantly enhance peptide quantification success rates (>97% for 400 targets, >95% for 200 difficult targets).
- Enabled a quantitative assay for 125 macroautophagy proteins with >95% success.
- Revealed differential macroautophagy protein expression across four human cell lines.
Conclusions:
- GoDigViewer and priming runs optimize GoDig assays for broader proteomic applications.
- This approach facilitates the study of complex proteomes and biological pathways.
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