Multi-in-One: Multiple-Proteases, One-Hour-Shot Strategy for Fast and High-Coverage Phosphoproteomic Investigation.

Area of Science:

• Proteomics
• Biochemistry
• Molecular Biology

Background:

• Traditional multiple-protease shotgun proteomics improves phosphosite identification but is time-consuming and lacks reproducibility.
• There is a need for faster, more reproducible methods to achieve comprehensive phosphosite coverage.

Purpose of the Study:

• To develop and validate a multi-in-one strategy for efficient and reproducible phosphosite identification and quantification.
• To reduce the time and complexity associated with traditional phosphoproteomics workflows.

Main Methods:

• A "three-in-one" workflow combining multiple-protease digestion, one-step enrichment, and one-shot data-independent acquisition (DIA).
• A "six-in-one" strategy utilizing a rapid 66-minute chromatographic method for phosphosite localization.
• Application of the strategy to identify phosphorylation events on low-abundance transcription factors in living cells.

Main Results:

• The "three-in-one" strategy identified over 19,700 and 13,500 phosphosites using trypsin-like and non-trypsin-like proteases, respectively.
• Nearly 30,000 phosphosites were quantified with high reproducibility using the combined strategy.
• The "six-in-one" strategy localized 19,445 phosphosites, significantly reducing database search time.
• Discovered 2,675 phosphorylation events on low-abundance transcription factors with high coverage.

Conclusions:

• The multi-in-one strategy offers a time-saving, sensitive, and highly reproducible method for large-scale phosphoproteomics.
• This approach significantly improves phosphosite coverage and enables discovery of phosphorylation events on low-abundance proteins.
• The developed strategy makes multi-protease digestion practical for extensive proteomic analyses.