Added PubMed Id 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.