Proteomic identification of cellular protease substrates using isobaric tags for relative and absolute quantification...

Area of Science:

• Biochemistry
• Proteomics
• Molecular Biology

Background:

• Protease activity is crucial for cellular functions and implicated in various diseases.
• Identifying protease substrates is key to understanding proteolysis's role in health and disease.
• Current methods may have limitations in identifying novel substrates in complex biological systems.

Purpose of the Study:

• To present a novel application of Isobaric tags for relative and absolute quantification (iTRAQ) for identifying protease substrates.
• To demonstrate the utility of iTRAQ in the cellular context for discovering new cleavage sites.
• To provide a method for understanding the functional impact of proteolysis on the proteome.

Main Methods:

• Utilized amine-targeted iTRAQ labeling on tryptic peptides from secreted proteins and shed domains.
• Applied a second iTRAQ tag to peptides from control cells for comparative analysis.
• Employed MS/MS fragmentation to sequence labeled peptides and identify unique signature ion peaks.

Main Results:

• Successfully labeled and quantified peptides originating from protease-transfected cells versus control cells.
• The unique signature ion peaks generated by MS/MS fragmentation enabled precise identification of labeled peptides.
• Comparison of peak areas allowed for relative quantification of identified protease substrates.

Conclusions:

• Isobaric tags for relative and absolute quantification (iTRAQ) offer a robust method for identifying protease substrates.
• This approach facilitates the study of normal and dysregulated proteolysis in various biological contexts.
• The findings contribute to a deeper understanding of protease function and its implications in disease.