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. 2023:2718:111-135.
doi: 10.1007/978-1-0716-3457-8_7.

Sensitive and High-Throughput Exploration of Protein N-Termini by TMT-TAILS N-Terminomics

Konstantinos Kalogeropoulos  1 , Louise Bundgaard  1 , Ulrich Auf dem Keller  2
Affiliations

Sensitive and High-Throughput Exploration of Protein N-Termini by TMT-TAILS N-Terminomics

Konstantinos Kalogeropoulos et al. Methods Mol Biol. 2023.

Abstract

Terminal amine isotopic labeling of substrates (TAILS) is a sensitive and robust quantitative mass spectrometry (MS)-based proteomics method used for the characterization of physiological or proteolytically processed protein N-termini, as well as other N-terminal posttranslational modifications (PTMs). TAILS is a well-established, high-throughput, negative enrichment workflow that enables system-wide exploration of N-terminomes independent of sample complexity. TAILS makes use of amine reactivity of free N-termini and a highly efficient aldehyde-functionalized polymer to deplete internal peptides generated after proteolytic digestion during sample preparation. Thereby, it enriches for natural N-termini, allowing for unbiased and complete investigation of differential proteolysis, protease substrate discovery, and analysis of N-terminal PTMs. In this chapter, we provide a state-of-the-art protocol, with detailed steps in all parts of the TAILS sample preparation, MS analysis, and post-processing of acquired data.

Keywords: Degradomics; Mass spectrometry-based proteomics; N-terminal posttranslational modifications; N-terminomics; Protease characterization; Protease substrate discovery; TAILS.

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