Extended Bottom-Up Proteomics with Secreted Aspartic Protease Sap9

We investigate the benefits and experimental feasibility of approaches enabling the shift from short (1.7 kDa on average) peptides in bottom-up proteomics to about twice longer (similar to 3.2 kDa on average) peptides in the so-called extended bottom-up proteomics. Candida albicans secreted aspartic protease Sap9 has been selected for evaluation as an extended bottom-up proteomic-grade enzyme due to its suggested dibasic cleavage specificity and ease of production. We report the extensive characterization of Sap9 specificity and selectivity revealing that protein cleavage by Sap9 most often occurs in the vicinity of proximal basic amino acids, and in select cases also at basic and hydrophobic residues. Sap9 is found to cleave a large variety of proteins in a relatively short, similar to 1 h, period of time and it is efficient in a broad pH range, including slightly acidic, e. g., pH 5.5, conditions. Importantly, the resulting peptide mixtures contain representative peptides primarily in the target 3-7 kDa range. The utility and advantages of this enzyme in routine analysis of protein mixtures are demonstrated and the limitations are discussed. Overall, Sap9 has a potential to become an enzyme of choice in an extended bottom-up proteomics, which is technically ready to complement the traditional bottom-up proteomics for improved targeted protein structural analysis and expanded proteome coverage. Biological significance Advances in biological applications of mass spectrometry-based bottom-up proteomics are oftentimes limited by the extreme complexity of biological samples, e.g., proteomes or protein complexes. One of the reasons for it is in the complexity of the mixtures of enzymatically (most often using trypsin) produced short (