Top-Down Proteomics and the Challenges of True Proteoform Characterization

Area of Science:

• Proteomics
• Biochemistry
• Molecular Biology

Background:

• Top-down proteomics (TDP) aims to identify intact protein forms (proteoforms).
• Characterizing proteoforms requires defining the base sequence and identifying mass shifts, localizing them to specific sites.
• Understanding proteoform roles is crucial for protein structure-function relationships and post-translational modification (PTM) analysis.

Purpose of the Study:

• To discuss the challenges in analyzing kindred proteoform populations.
• To highlight the difficulties in annotating PTMs on average protein representations.
• To outline technical requirements for high-quality fragmentation data for site-specific PTM definition.

Main Methods:

• Perspective-based discussion on TDP challenges.
• Focus on annotation of PTMs in complex proteoform mixtures.
• Consideration of technical requirements for mass spectrometry (MS) data acquisition.

Main Results:

• Characterization of heavily modified proteins (>~30 kDa) is problematic, especially within kindred proteoform populations.
• Increased PTMs compound the challenge due to numerous theoretical combinations.
• Time-intensive separation of proteoforms is often necessary before MS analysis.

Conclusions:

• Defining and pinpointing PTMs on intact proteins, particularly in complex mixtures, remains a significant challenge in TDP.
• Robust site-specific PTM definition requires high-quality fragmentation data and efficient proteoform separation.
• Current TDP methods face limitations in fully elucidating complex proteoform profiles and their unique roles.