# Proteome-Wide Analysis and Surface Protein Isolation for Secretome Characterization Reveal Insights into the Biology of the Leaf-Cutter Ant Acromyrmex echinatior

## TL;DR

This study uses proteomic techniques to analyze the internal and external proteins of leaf-cutter ants, revealing age-related changes and functions related to symbiosis and environmental interactions.

## Contribution

A dual-layered proteomic approach and acid-based extraction method were developed to characterize the whole-body and secreted cuticular proteomes of Acromyrmex echinatior.

## Key findings

- 4,428 proteins were quantified across four adult ant ages, revealing clusters related to muscle development, lipid metabolism, and immune responses.
- 323 secreted cuticular proteins were identified, many associated with stress response, microbial defense, and cuticle sclerotization.
- Tropomyosin-family proteins were highly enriched in the secretome and showed significant temporal abundance changes.

## Abstract

Characterizing the proteome of an organism can provide
critical
insights into the proteins that regulate key biological processes
such as development, physiology, and environmental interactions. While
proteome-wide analyses reveal broad protein dynamics, spatially resolved
approaches can uncover specific, localized functions. For example,
the leaf-cutter ant Acromyrmex echinatior secretes a unique protein layer that coats its exoskeleton and interacts
with biotic and abiotic factors, including its symbiotic bacterium Pseudonocardia. In this study, to characterize both
the whole-body proteome and the externally secreted cuticular protein
layer of A. echinatior, we utilize
a dual-layered proteomic approach. Using diaPASEF, we quantified 4,428
proteins across four early adult ages, uncovering distinct age-dependent
protein clusters enriched in muscle development, lipid metabolism,
and immune-related responses. We then developed an acid-based extraction
method to isolate the externally secreted protein layer, identifying
323 secreted proteins via the ddaPASEF acquisition. Many of these
proteins exhibited temporal abundance changes and were associated
with functions, such as environmental stress response, microbial defense,
and cuticle sclerotization. Notably, tropomyosin-family proteins were
highly enriched in the external secretome and exhibited significant
changes across early adult time points, potentially linking these
ion-binding molecules to metal-enrichment processes occurring during
this crucial stage. Together, this work reveals dynamic changes in
the internal and surface proteomes of young adult A.
echinatior ants and provides a methodological framework
for further probing localized extra-cuticular protein function in
complex biological systems.