PBC, an easy and efficient strategy for high-throughput protein C-terminome profiling

Area of Science:

• Proteomics
• Biochemistry
• Molecular Biology

Background:

• High-throughput profiling of protein C-termini remains challenging.
• Existing C-terminal peptide enrichment methods are costly, time-consuming, and inefficient.
• Proteomics offers a powerful approach for systematic C-termini studies.

Purpose of the Study:

• To develop a novel, efficient, and high-throughput strategy for C-terminomics analysis.
• To overcome limitations of current chemical derivatization-based C-terminomics methods.
• To enable comprehensive investigation of protein C-terminome, including degradomics and PTMs.

Main Methods:

• Developed a 2-pyridinecarboxaldehyde (2-PCA) and biotin labeling-based C-terminomic (PBC) strategy.
• Utilized the reaction selectivity of 2-PCA with N-terminal α-amino groups.
• Leveraged the high affinity between biotin and streptavidin for enrichment.
• Performed triplicate PBC experiments on 293 T cell line.

Main Results:

• Identified 1,975 C-terminal peptides from 1,190 proteins, a 180% increase compared to previous studies.
• Achieved the highest reported enrichment efficiency (68%) for C-terminomics methods.
• Uncovered 50 proteins with truncated C-termini, enriched in extracellular vesicles and ribosomes.
• Systematically characterized C-terminal post-translational modifications (PTMs) in 293 T cells.

Conclusions:

• The PBC strategy is a powerful tool for high-efficiency and high-throughput C-terminome analysis.
• PBC facilitates protein C-terminal degradomics and PTMs investigation.
• This method significantly advances the field of C-terminomics research.