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

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.