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

Global characterization of protein N termini provides valuable information on proteome dynamics and diversity in health and disease. Driven by the progress in mass spectrometry-based proteomics, novel approaches for the dedicated investigation of protein N termini and protease substrates have been recently developed. Terminal amine isotopic labeling of substrates (TAILS) is a quantitative proteomics approach suitable for high-throughput and system-wide profiling of protein N termini in complex biological matrices. TAILS employs isotopic labeling of primary amines of intact proteins in combination with an amine-reactive high molecular weight polymer (HPG-ALD) for depletion of internal tryptic peptides and high enrichment of protein N termini by negative selection. Thereby, TAILS allows simultaneous identification of the natural N termini, protease-generated neo-N termini, and endogenously modified (e.g., acetylated) N termini. In this chapter, we provide a protocol for tandem mass tag (TMT)-TAILS analysis and further discuss specific considerations regarding N-terminome data interpretation using Proteome Discoverer™ software.