ExteNDing Proteome Coverage with Legumain as a Highly Specific Digestion Protease

Area of Science:

• Proteomics
• Enzymology
• Biochemistry

Background:

• Bottom-up mass spectrometry relies on proteolytic enzymes for peptide generation.
• Trypsin is the most common enzyme, but alternative proteases are needed for comprehensive coverage.
• Identifying peptides not accessible by trypsin is crucial for deeper proteome analysis.

Purpose of the Study:

• To investigate the utility of human legumain as a protease for bottom-up mass spectrometry.
• To assess legumain's specificity and its ability to enhance proteome and protein sequence coverage.
• To explore legumain's application in identifying protein N-termini and N-glycosylation sites.

Main Methods:

• In-solution digestion of proteomes from *Escherichia coli*, mouse embryonic fibroblasts, and *Arabidopsis thaliana* using legumain.
• Comparison of legumain's digestion efficiency and peptide complementarity with trypsin and GluC.
• Sequential sample treatments with legumain and PNGaseF for N-glycosylation site analysis.

Main Results:

• Legumain demonstrated strict specificity for cleavage after asparagine and aspartic acid residues.
• Legumain generated peptides complementary to trypsin, increasing overall proteome and sequence coverage.
• Legumain enabled identification of protein N-termini missed by trypsin and GluC.
• Legumain facilitated robust identification and validation of N-glycosylation sites.

Conclusions:

• Human legumain is a practical and efficient protease for expanding proteome coverage in mass spectrometry.
• Legumain offers unique advantages for characterizing protein N-termini and post-translational modifications, particularly N-glycosylation.
• The findings highlight legumain's potential to complement trypsin in comprehensive proteomic studies.