RESUMO
The colonial ascidian Boytryllus schlosseri is an invasive marine chordate that thrives under conditions of anthropogenic climate change. We show that the B. schlosseri expressed proteome contains unusually high levels of proteins that are adducted with 4-hydroxy-2-nonenal (HNE). HNE represents a prominent posttranslational modification resulting from oxidative stress. Although numerous studies have assessed oxidative stress in marine organisms HNE protein modification has not previously been determined in any marine species. LC/MS proteomics was used to identify 1052 HNE adducted proteins in B. schlosseri field and laboratory populations. Adducted amino acid residues were ascertained for 1849 modified sites, of which 1195 had a maximum amino acid localization score. Most HNE modifications were at less reactive lysines (rather than more reactive cysteines). HNE prevelance on most sites was high. These observations suggest that B. schlosseri experiences and tolerates high intracellular reactive oxygen species levels, resulting in substantial lipid peroxidation. HNE adducted B. schlosseri proteins show enrichment in mitochondrial, proteostasis, and cytoskeletal functions. Based on these results we propose that redox signaling contributes to regulating energy metabolism, the blastogenic cycle, oxidative burst defenses, and cytoskeleton dynamics during B. schlosseri development and physiology. A DIA assay library was constructed to quantify HNE adduction at 72 sites across 60 proteins that represent a holistic network of functionally discernable oxidative stress bioindicators. We conclude that the vast amount of HNE protein adduction in this circumpolar tunicate is indicative of high oxidative stress tolerance contributing to its range expansion into diverse environments. NEW & NOTEWORTHY: Oxidative stress results from environmental challenges that increase in frequency and severity during the Anthropocene. Oxygen radical attack causes lipid peroxidation leading to HNE production. Proteome-wide HNE adduction is highly prevalent in Botryllus schlosseri , a widely distributed, highly invasive, and economically important biofouling ascidian and the first marine species to be analyzed for proteome HNE modification. HNE adduction of specific proteins physiologically sequesters reactive oxygen species, which enhances fitness and resilience during environmental change.
RESUMO
Botryllus schlosseri, is a model marine invertebrate for studying immunity, regeneration, and stress-induced evolution. Conditions for validating its predicted proteome were optimized using nanoElute® 2 deep-coverage LCMS, revealing up to 4930 protein groups and 20,984 unique peptides per sample. Spectral libraries were generated and filtered to remove interferences, low-quality transitions, and only retain proteins with >3 unique peptides. The resulting DIA assay library enabled label-free quantitation of 3426 protein groups represented by 22,593 unique peptides. Quantitative comparisons of single systems from a laboratory-raised with two field-collected populations revealed (1) a more unique proteome in the laboratory-raised population, and (2) proteins with high/low individual variabilities in each population. DNA repair/replication, ion transport, and intracellular signaling processes were distinct in laboratory-cultured colonies. Spliceosome and Wnt signaling proteins were the least variable (highly functionally constrained) in all populations. In conclusion, we present the first colonial tunicate's deep quantitative proteome analysis, identifying functional protein clusters associated with laboratory conditions, different habitats, and strong versus relaxed abundance constraints. These results empower research on B. schlosseri with proteomics resources and enable quantitative molecular phenotyping of changes associated with transfer from in situ to ex situ and from in vivo to in vitro culture conditions.