Inserting Pre-analytical Chromatographic Priming Runs Significantly Improves Targeted Pathway Proteomics with Sample...

Area of Science:

• Proteomics
• Mass Spectrometry
• Computational Biology

Background:

• GoDig is a targeted pathway proteomics platform using tandem mass tags for high throughput.
• Current GoDig limitations include lower peptide-level success rates for challenging targets.

Purpose of the Study:

• To develop software (GoDigViewer) for visualizing GoDig runs to guide assay optimization.
• To improve peptide and protein quantification success rates in targeted proteomics.

Main Methods:

• Development of GoDigViewer software for detailed visualization of GoDig runs.
• Implementation of a new acquisition mode, "priming runs," involving multiple chromatographic runs before analysis.
• Application of priming runs for quantitative assays, including macroautophagy proteins.

Main Results:

• GoDigViewer facilitated the development of priming runs, enhancing accuracy and precision.
• Priming runs achieved >97% success for 400 peptide targets and >95% for 200 previously unquantified targets.
• A quantitative assay for 125 macroautophagy proteins was established with >95% success, revealing cell line-specific expression differences.

Conclusions:

• GoDigViewer and priming runs significantly enhance the performance of targeted proteomics assays.
• This approach enables robust quantification of previously intractable peptides and proteins.
• The method successfully profiled macroautophagy differences across human cell lines, demonstrating its utility in biological discovery.