Quantitative Multiplex Substrate Profiling of Peptidases by Mass Spectrometry

Area of Science:

• Biochemistry
• Proteomics
• Enzymology

Background:

• Proteolysis is crucial for biological processes and implicated in diseases.
• Understanding peptidase function requires tools to determine substrate specificity and cleavage efficiency.

Purpose of the Study:

• To develop and validate a quantitative assay for profiling peptidase activity.
• To apply the assay to characterize novel peptidases and complex biological samples.

Main Methods:

• Quantitative Multiplex Substrate Profiling by Mass Spectrometry (qMSP-MS) combines tandem mass tags (TMTs) with a peptide cleavage assay.
• The assay was validated using papain to determine cleavage efficiency for 275 peptide bonds.
• Unmodified synthetic peptides were used to assess both endo- and exo-peptidase activity.

Main Results:

• qMSP-MS successfully determined cleavage efficiency values for papain.
• The assay uncovered substrate specificity for intramembrane rhomboid peptidases.
• Hundreds of proteolytic activities were identified in complex biological samples, including lung cancer cell line secretions.
• Peptide substrates were ranked by turnover rate, enabling the design of reporter substrates.

Conclusions:

• qMSP-MS is a versatile tool for characterizing peptidase activity and substrate specificity.
• The assay can identify proteolytic activities in complex biological systems and disease-relevant samples.
• qMSP-MS has the potential to guide the development of targeted peptidase-activating drugs for cancer and infectious diseases.