Exploring Extracellular Matrix Degradomes by TMT-TAILS N-Terminomics.

Area of Science:

• Proteomics
• Biochemistry
• Mass Spectrometry

Background:

• Global characterization of protein N termini is crucial for understanding proteome dynamics in health and disease.
• Advancements in mass spectrometry-based proteomics have enabled new methods for investigating protein N termini and protease substrates.

Purpose of the Study:

• To present a detailed protocol for Tandem Mass Tag (TMT)-Terminal amine isotopic labeling of substrates (TAILS) analysis.
• To discuss considerations for interpreting N-terminome data obtained using Proteome Discoverer™ software.

Main Methods:

• Terminal amine isotopic labeling of substrates (TAILS) utilizes isotopic labeling of primary amines and an amine-reactive polymer (HPG-ALD).
• This method achieves depletion of internal tryptic peptides and enrichment of protein N termini via negative selection.
• The protocol details TMT-TAILS analysis for quantitative profiling.

Main Results:

• TAILS allows for the simultaneous identification of natural N termini, protease-generated neo-N termini, and modified N termini (e.g., acetylation).
• The method is suitable for high-throughput, system-wide profiling of protein N termini in complex biological matrices.

Conclusions:

• TMT-TAILS provides a robust quantitative proteomics approach for comprehensive N-terminome analysis.
• This method facilitates a deeper understanding of proteome dynamics and diversity in various biological states.