The first proteomics analysis of a colonial tunicate (Botryllus schlosseri) was carried out to support development of a DIA assay library and quantitative proteomics for this species. Deep proteome coverage (up to 4930 protein groups and 20,984 unique peptides in a single sample) was achieved, which enabled construction of a QC filtered DIA assay library consisting of nearly 4000 proteins that are represented by at least 3 unique peptides, each by at least 4 transitions. This library was used to compare protein abundances of two field populations and one lab-reared B. schlosseri population. Relative protein abundance data indicate that lab-rearing leads to greater proteome changes than those observed between distantly located field populations in Southern California and Northern Washington. STRING analysis revealed functions that are enriched in specific populations and proteins that are least and most variable within a given population.

Conditions for B. schlosseri quantitative proteomics were first optimized by testing different sample homogenization, LC gradients, and separation times. Three populations (two field, one lab) were analyzed to 1) establish an experimentally validated reference proteome, 2) reveal proteome differences between lab and field populations that originated from the same site, 3) reveal proteome differences between Northern (Tacoma, Washington) and Southern (Santa Barbara, California) populations, and 4) identify proteins that are most and least variable within a given population.

Samples were collected from two field sites and harvested from one population that was initially sexually reproduced and then asexually propagated under laboratory culture conditions. The two field sites were Tacoma, Washington (T) and Santa Barbara, California (W). The lab culture population was grown at the University of California, Santa Barbara (C). Samples consisted of single systems (flower-shaped colonies) of B. schlosseri. Numbers of biological replicates used in this study were 12 (W), 12 (C), and 20 (T) samples.