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Extended bottom-up proteomics with secreted aspartic protease Sap9
Ünige A Laskay1, Kristina Srzentić1, Michel Monod2
1Biomolecular Mass Spectrometry Laboratory, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland.
Journal of Proteomics
|August 16, 2014
Summary
Extended bottom-up proteomics uses longer peptides (~3.2 kDa) for improved proteome analysis. Candida albicans Sap9 enzyme shows potential for producing these longer peptides, enhancing protein structural analysis and proteome coverage.
Area of Science:
- Proteomics
- Biochemistry
- Enzymology
Background:
- Bottom-up proteomics, typically using trypsin, produces short peptides (<3 kDa) that can complicate analysis of complex biological samples.
- The complexity of short peptide mixtures can limit liquid chromatography and mass spectrometry capabilities, affecting protein modification localization.
- Advances in mass spectrometry and chromatography offer opportunities to improve proteome analysis by increasing peptide size.
Purpose of the Study:
- To investigate the benefits and experimental feasibility of extended bottom-up proteomics, which utilizes longer peptides (~3.2 kDa).
- To evaluate Candida albicans secreted aspartic protease Sap9 as a potential enzyme for generating these longer peptides.
- To characterize the specificity, selectivity, and efficiency of Sap9 for proteomic applications.
Main Methods:
- Selected Candida albicans secreted aspartic protease Sap9 for its dibasic cleavage specificity and ease of production.
- Extensively characterized Sap9 specificity and selectivity through protein cleavage experiments.
- Assessed Sap9 efficiency across a broad pH range and its ability to cleave various proteins within a short timeframe.
Main Results:
- Sap9 cleavage primarily occurs near proximal basic amino acids, with occasional cleavage at basic and hydrophobic residues.
- Sap9 efficiently cleaves a diverse range of proteins within approximately 1 hour.
- The enzyme functions effectively across a broad pH range, including slightly acidic conditions (pH 5.5).
- Resulting peptide mixtures predominantly feature peptides in the desired 3-7 kDa range.
Conclusions:
- Sap9 demonstrates potential as a proteomic-grade enzyme for extended bottom-up proteomics.
- This approach can complement traditional bottom-up proteomics by enabling improved targeted protein structural analysis.
- Extended bottom-up proteomics, facilitated by enzymes like Sap9, offers expanded proteome coverage and addresses limitations of short peptide analysis.
