Abstract

ABSTRACT OF THE DISSERTATION Development of a Quantitative Top-Down Proteomics Platform for Arginylated Proteoform Analysis by Richard Myer Searfoss Doctor of Philosophy in Biology and Biomedical Sciences Biochemistry, Biophysics, & Structural Biology Washington University in St. Louis, 2026 Professor Benjamin A. Garcia, Chair Top-down proteomics is a technique that utilizes mass spectrometry to identify and quantify biologically relevant proteoforms, or proteins with unique amino acid sequence and post-translational modification (PTM) signature. Challenges that exist in top-down proteomics include localization of PTMs, protein separation, and signal dilution. Here, we developed a top-down proteomics platform on a time-of-flight mass spectrometer that takes advantage of microflow liquid chromatography for improved protein separation, and electron-activated dissociation for enhanced protein fragmentation and site-localization. This platform was optimized with recombinant proteins and was demonstrated to analyze proteins as large as intact antibodies. We localized the exact sites of various PTMs with sequence coverage reaching 50-95%. After successful development of this platform, we sought to apply it to characterizing arginylated proteoforms. Arginylation is a PTM where an arginine residue is conjugated onto the N-terminus of a protein primarily to signal it for degradation, as well as fulfilling other biological roles. Given that this modification exactly mimics the translational inclusion of an arginine residue into a protein, it has been challenging to study. We applied our top-down proteomics platform to characterizing a known substrate of the arginylation pathway, calreticulin. We captured in vitro and in vivo arginylation, quantified unmodified versus arginylated proteoforms, and demonstrated the utility of isotopically-labeled arginine to identify and quantify arginylated substrates. We further applied this platform to characterizing other arginylated substrates including SSBP1 and ERO1A. With a working platform, we last monitored the arginylation of a β-actin fusion construct with a variable N-terminal residue. Following conversion of the N-terminal residue into a proper substrate for arginylation, clear differences in arginylation efficiency and preference by the ATE1 enzyme were identified. This is the first such exploration of arginylation on intact proteins with mass spectrometry and has contributed a novel platform to the field for studying a challenging PTM.

Committee Chair

Benjamin Garcia

Committee Members

Elizabeth Draganova; Michael Gross; Michael Major; Young Goo

Degree

Doctor of Philosophy (PhD)

Author's Department

Biology & Biomedical Sciences (Biochemistry)

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

4-24-2026

Language

English (en)

Author's ORCID

https://orcid.org/0000-0001-6235-2595

Download

Available for download on Thursday, October 22, 2026

Included in

Biochemistry Commons

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