Validation of Top-Down Proteomics Data by Bottom-Up-Based N-Terminomics Reveals Pitfalls in Top-Down-Based...

Area of Science:

• Proteomics
• Biochemistry
• Molecular Biology

Background:

• Bottom-up proteomics (BUP) is standard for N-terminomics, focusing solely on N-terminal peptides.
• Top-down proteomics (TDP) offers potential for broader proteoform characterization, including N- and C-termini and post-translational modifications.

Purpose of the Study:

• To evaluate the potential and limitations of TDP for terminomics.
• To compare TDP-identified N-termini with established BUP N-terminomics approaches.

Main Methods:

• Two established TDP workflows were applied to Caenorhabditis elegans proteome analysis.
• N-termini identified by TDP were validated using a BUP-based N-terminomics approach.
• Reductive dimethylation was explored in TDP for N-termini confirmation.

Main Results:

• TDP workflows identified 1658 proteoforms.
• Only 25% of TDP-identified N-termini were successfully verified by BUP.
• Limitations include trypsin's inability to detect certain N-termini in BUP and artifactual termini in TDP.

Conclusions:

• TDP holds promise for terminomics but currently has significant limitations.
• Methodological challenges in both BUP and TDP contribute to low overlap and verification rates.
• Future developments in data quality control are needed to improve TDP's utility for protein termini detection.