Abstract
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.
| Original language | English |
|---|---|
| Title of host publication | Collagen |
| Editors | Irit Sagi, Nikolaos A. Afratis |
| Number of pages | 12 |
| Publisher | Humana Press |
| Publication date | 2019 |
| Pages | 115-126 |
| Chapter | 8 |
| ISBN (Print) | 978-1-4939-9094-8 |
| ISBN (Electronic) | 978-1-4939-9095-5 |
| DOIs | |
| Publication status | Published - 2019 |
| Series | Methods in Molecular Biology |
|---|---|
| Volume | 1944 |
| ISSN | 1064-3745 |
Keywords
- N-terminome
- Proteolysis
- Proteome Discoverer™
- TAILS
- TMT
Cite this
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Exploring extracellular matrix degradomes by TMT-TAILS N-terminomics. / Madzharova, Elizabeta; Sabino, Fabio; auf dem Keller, Ulrich.
Collagen. ed. / Irit Sagi; Nikolaos A. Afratis. Humana Press, 2019. p. 115-126 (Methods in Molecular Biology, Vol. 1944).Research output: Chapter in Book/Report/Conference proceeding › Book chapter › Research › peer-review
TY - CHAP
T1 - Exploring extracellular matrix degradomes by TMT-TAILS N-terminomics
AU - Madzharova, Elizabeta
AU - Sabino, Fabio
AU - auf dem Keller, Ulrich
PY - 2019
Y1 - 2019
N2 - 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.
AB - 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.
KW - N-terminome
KW - Proteolysis
KW - Proteome Discoverer™
KW - TAILS
KW - TMT
U2 - 10.1007/978-1-4939-9095-5_8
DO - 10.1007/978-1-4939-9095-5_8
M3 - Book chapter
SN - 978-1-4939-9094-8
SP - 115
EP - 126
BT - Collagen
A2 - Sagi, Irit
A2 - Afratis, Nikolaos A.
PB - Humana Press
ER -