Probing the efficiency of proteolytic events by positional proteomics

Area of Science:

• Biochemistry
• Proteomics
• Molecular Biology

Background:

• Mass spectrometry techniques identify protease substrates and specificities.
• Distinguishing critical from bystander substrates requires cleavage efficiency data.
• Protease function understanding relies on identifying physiologically relevant substrates.

Purpose of the Study:

• To develop and apply a quantitative mass spectrometry method to measure protease cleavage efficiency.
• To identify and categorize substrates of human granzyme B (hGrB) based on cleavage efficiency.
• To identify potential regulators of hGrB-induced apoptosis.

Main Methods:

• Quantitative mass spectrometry with SILAC (Stable Isotope Labeling by Amino acids in Cell culture).
• Isolation of N-terminal peptides using Combined Fractional Diagonal Chromatography (COFRADIC).
• Treatment of Jurkat cell lysate with hGrB across different incubation periods.

Main Results:

• Identified 101 unique hGrB-specific neo-N-termini in 86 proteins.
• Quantified cleavage efficiency for numerous hGrB substrates in vitro.
• Confirmed known substrate Bid and identified potential apoptosis regulators Bnip2 and Akap-8 as efficient hGrB substrates.

Conclusions:

• Quantitative proteomics effectively distinguishes efficient protease cleavage sites.
• This method aids in identifying physiologically relevant protease substrates.
• Identified novel substrates of hGrB, potentially involved in apoptosis regulation.