Top-down Proteomics for the Characterization and Quantification of Calreticulin Arginylation

Area of Science:

• Biochemistry
• Proteomics
• Molecular Biology

Background:

• Arginylation, catalyzed by arginyltransferase 1 (ATE1), involves adding arginine to proteins and is vital for cellular processes.
• Loss of arginylation in mouse models leads to severe developmental defects, highlighting its biological significance.
• Current bottom-up proteomics methods can identify arginylation sites but struggle with quantitative proteoform analysis.

Purpose of the Study:

• To develop and validate a top-down proteomics workflow for characterizing and quantifying protein arginylation.
• To apply this workflow to analyze arginylation of calreticulin (CALR).
• To compare fragmentation techniques for optimal analysis of modified proteoforms.

Main Methods:

• Developed a top-down proteomics approach for arginylation analysis.
• Generated fully arginylated calreticulin (R-CALR) by genetic modification.
• Utilized liquid chromatography-mass spectrometry (LC-MS) with electron-activated dissociation (EAD) and ultraviolet photodissociation (UVPD).

Main Results:

• The top-down method successfully characterized and quantified CALR arginylation.
• EAD provided N-terminal fragmentation for site localization, while UVPD enhanced sequence coverage in challenging domains.
• The workflow demonstrated the ability to identify and quantify CALR arginylation at various levels (absent, endogenous, high).

Conclusions:

• This work presents the first application of top-down proteomics for characterizing post-translational arginylation.
• The developed method enables precise identification and quantification of arginylated proteoforms.
• This approach advances the study of arginylation's role in biological systems.