Sensitive Determination of Proteolytic Proteoforms in Limited Microscale Proteome Samples.

Area of Science:

• Proteomics
• Biochemistry
• Molecular Biology

Background:

• Protein N termini are crucial for identifying proteoforms and disease perturbations.
• Current N-terminal peptide enrichment methods require large sample amounts, limiting analysis of microscale samples.
• Many proteolytic processes occur in limited time and space, necessitating sensitive enrichment techniques.

Purpose of the Study:

• To present High-efficiency Undecanal-based N Termini EnRichment (HUNTER), a novel method for N-terminal peptide enrichment.
• To enable proteome-wide N-terminome analysis from microscale samples.
• To facilitate the study of proteolytic processing and protease substrates in challenging biological contexts.

Main Methods:

• Development of the HUNTER method utilizing undecanal for selective N-terminal peptide enrichment.
• Application of HUNTER to analyze N termini from minute amounts of HeLa cell lysate.
• Demonstration of HUNTER's broad applicability on human primary immune cells, mitochondrial fractions, and *Arabidopsis thaliana* samples.

Main Results:

• HUNTER successfully identified over 1000 N termini from as little as 2 μg of raw HeLa cell lysate.
• The first N-terminome analysis of sorted human primary immune cells and pediatric cancer patient mitochondrial fractions was achieved.
• Protease substrate identification was performed on individual *Arabidopsis thaliana* seedlings, including mutant lines.
• The HUNTER workflow was automated on a liquid handling system for clinical degradomics applications.

Conclusions:

• HUNTER is a robust, sensitive, and scalable method for N-terminome analysis of microscale samples.
• The method significantly expands the scope of proteomic and degradomic research, particularly in clinical and plant biology.
• Automated HUNTER processing demonstrates feasibility for clinical degradomics using liquid biopsies.