Profiling of Protein N-Termini and Their Modifications in Complex Samples

Area of Science:

• Proteomics
• Biochemistry
• Molecular Biology

Background:

• Protein N termini provide insights into proteome functional states, including translation, modification, and proteolytic processing.
• N termini serve as direct indicators of proteolytic activity, aiding in the identification of protease substrates and their biological functions.

Purpose of the Study:

• To describe the Terminal Amine Isotope Labeling of Substrates (TAILS) protocol for enriching protein N-terminal peptides.
• To enable simultaneous enrichment of genome-encoded, protease-generated, and modified N termini from complex proteomic samples.

Main Methods:

• Utilized the TAILS technique for negative selection-based enrichment of protein N-terminal peptides.
• Applied mass spectrometry for comprehensive profiling of N termini and their modifications.
• Developed a TAILS-compatible protocol for plant proteome preparation.

Main Results:

• Successfully enriched diverse protein N termini, including neo-N termini and modified termini.
• Enabled single-experiment profiling of all protein N termini and associated modifications.
• Provided a detailed protocol for plant N-terminome analysis.

Conclusions:

• TAILS is an effective method for comprehensive N-terminome profiling.
• The protocol facilitates the study of proteolytic regulation and protease substrate identification.
• Detailed guidelines for plant N-terminome data analysis and annotation are provided.