Development of a Protein Mass Spectrometric Assay for the Characterization of Colorectal Cancer Exosomes

Authors

• Frank J. Reed IV, Belmont UniversityFollow

Publication Date

Fall 11-23-2026

College

College of Pharmacy & Health Sciences

SURS Faculty Advisor

Amy Ham

Presentation Type

Poster Presentation

Abstract

Exosomes (sEV) carry proteins involved in intracellular communication and colorectal cancer (CRC) biology, but their morphologic overlap with non-vesicular extracellular particles (NVEPs), and other extracellular vesicles (EVs), including exomeres and supermeres, complicates proteomic characterization. This aim of this study is to develop mass spectrometric assays that evaluate sEV enrichment, purity, and distinguish sEV CRC-associated protein cargo from EV and NVEP CRC-associated protein cargo to better classify the role of sEVs in CRC biology.

Our lab previously analyzed conditioned media from DiFi, cystic clone (CC), and spikey clone (SC) CRC cell lines purified using dialysis, concentration, and electrophoresis enrichment (DCE) by LC-MS/MS. Comparison with datasets generated using ultracentrifugation (UC), a widely used reference method for sEV isolation, identified 656 shared proteins, with 281 uniquely detected from DCE, and 47 uniquely detected from UC, highlighting the need to further characterize co-isolated material. Among the identified proteins were several previously reported to be enriched in sEVs, exomeres, or supermeres, providing potential targets for parallel reaction monitoring (PRM) assay development.  Candidate proteins and their corresponding numbers of identified distinct peptides included sEV-associated ALIX (41), CD73 (6), FLOT1 (4), FLOT2 (5), TSG101 (8), CD81 (7), and CD63 (4); exomere-associated VCP (38), FASN (72), ACLY (21), and PKM (33); and supermere-associated ENO1 (14), ALDOA (28), HSP8 (34), and TGFBI (23). EGFR (56), previously reported to be associated with sEVs, was also identified as a candidate of particular interest given its elevated expression in certain CRC cell lines, including DiFi. Identified peptides for these and other proteins are currently being evaluated for sequence uniqueness, potential for missed cleavages, spectral consistency, charge-state suitability, and susceptibility to variable modifications such as oxidation and phosphorylation, to prioritize reliable targets for PRM assay development.

Following evaluation and selection of candidate peptides, a label-free PRM assay will be developed and applied to newly isolated samples to compare protein signals across CRC cell lines and purification methods. By targeting proteins previously associated with sEVs, exomeres, supermeres, and CRC, this approach aims to evaluate their utility as markers for distinguishing extracellular particle populations, assessing the relative purity of isolated preparations, and improving the reproducibility of proteomic analyses.

Recommended Citation

Reed, Frank J. IV, "Development of a Protein Mass Spectrometric Assay for the Characterization of Colorectal Cancer Exosomes" (2026). Science University Research Symposium (SURS). 337.
https://repository.belmont.edu/surs/337