Expanding proteome coverage with orthogonal-specificity α-lytic proteases

Area of Science:

• Proteomics
• Biochemistry
• Mass Spectrometry

Background:

• Traditional bottom-up proteomics relies on trypsin for protein digestion, but this often results in incomplete proteome coverage.
• Existing alternative proteases primarily target charged amino acid residues, limiting their ability to complement trypsin's specificity.

Purpose of the Study:

• To evaluate the utility of two novel proteases, wild type α-lytic protease (WaLP) and M190A α-lytic protease (MaLP), for enhancing proteome coverage in bottom-up proteomics.
• To assess the impact of these proteases on peptide characteristics and their ability to identify novel post-translational modifications.

Main Methods:

• Bottom-up proteomics was performed using separate digestions with trypsin, LysC, WaLP, and MaLP.
• Mass spectrometry (MS/MS) was employed to analyze peptide fragmentation, length, and yield.
• Protease specificity for aliphatic amino acid side chains was investigated.

Main Results:

• Combining data from trypsin, LysC, WaLP, and MaLP digestions increased proteome coverage by 101% compared to trypsin alone.
• Novel phosphorylation sites were identified in the Schizosaccharomyces pombe proteome, including sites on peptides too short for tryptic identification or in non-tryptic sequences.
• Coverage of membrane proteins increased by 350% when using the combined protease approach.

Conclusions:

• WaLP and MaLP are effective novel proteases for improving proteome coverage in bottom-up proteomics.
• The enhanced coverage facilitates the identification of previously undetected post-translational modifications and improves the analysis of challenging protein types like membrane proteins.