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Published on: October 27, 2023
Evaluation of the low-specificity protease elastase for large-scale phosphoproteome analysis
Bin Wang1, Rainer Malik, Erich A Nigg
1Department of Cell Biology, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.
Analytical Chemistry
|November 15, 2008
Summary
Using elastase alongside trypsin significantly enhances phosphorylation site mapping in phosphoproteome studies. This complementary enzyme approach nearly doubles detected sites, revealing unique phosphorylation events for comprehensive proteome analysis.
Area of Science:
- Proteomics
- Cell Biology
- Biochemistry
Background:
- Comprehensive phosphorylation site mapping is crucial for phosphoproteome studies.
- Achieving complete protein sequence coverage with a single protease is challenging.
Purpose of the Study:
- To evaluate elastase's utility in increasing phosphorylation site coverage compared to trypsin.
- To enhance phosphoproteome analysis of mitotic spindle proteins.
Main Methods:
- Utilized high-accuracy Orbitrap mass spectrometry.
- Analyzed elastase cleavage preferences for optimized database searching.
- Employed a dedicated two-step database search strategy.
- Compared elastase digestion with traditional trypsin digestion.
Main Results:
- Elastase digestion approximately doubled the number of detectable phosphorylation sites.
- Phosphorylation sites identified by trypsin and elastase showed less than 10% overlap, indicating high complementarity.
- Identified 1068 phosphorylation sites with trypsin and 467 with elastase.
- 30% of identified phosphorylation sites were exclusively found after elastase digestion.
Conclusions:
- Elastase is a valuable enzyme for enhancing phosphorylation site coverage in phosphoproteome studies.
- Combining elastase with trypsin provides complementary data, leading to more comprehensive proteome analysis.
- This dual-enzyme approach significantly improves the depth of phosphoproteome profiling.
