Top-Down Proteomic Characterization of Truncated Proteoforms

Area of Science:

• Proteomics
• Molecular Biology
• Cell Biology

Background:

• Proteolytic cleavage generates diverse protein forms.
• Identifying truncated proteins is challenging without enrichment methods.
• Extracellular vesicles from myeloid-derived suppressor cells play roles in immunosuppression.

Purpose of the Study:

• To develop and validate a top-down proteomic strategy for global identification of truncated proteins.
• To characterize truncated proteoforms in immunosuppressive extracellular vesicles.
• To identify potential proteases responsible for protein truncation.

Main Methods:

• Top-down proteomics with semiautomated data analysis.
• Analysis of extracellular vesicles from myeloid-derived suppressor cells.
• Identification of N- and C-terminally truncated proteoforms and internal polypeptides.

Main Results:

• Successfully identified over 1000 truncated proteoforms.
• Demonstrated the ability to identify proteins truncated at both termini, one terminus, or internal fragments.
• Derived binding motifs from identified proteoforms to infer protease activity.

Conclusions:

• The developed proteomic strategy is effective for comprehensive analysis of protein truncation.
• This approach facilitates the study of protein processing in biological systems, such as immunosuppressive extracellular vesicles.
• The findings provide insights into the proteases involved in generating truncated protein variants.