Isolation of phosphoproteins from symbiotic and aposymbiotic Aiptasia anemones for elucidation of the phosphoproteome v1

The analysis of gene expression data via RNA sequencing (RNA-Seq) has become a standard method for model and non-model organisms to identify genes of interest associated with a specific stressor of physiological state. More recently, mass spectrometry (MS)-based proteomics started to become a standard method to assay the phenotype of an organism via means of its expressed proteins. MS has also become the method of choice for the study of protein phosphorylation. Phosphoproteomics targets proteins that are subjected to phosphorylation via means of their serine, threonine, and tyrosine residues. Phosphorylation is a reversible modification that plays a key role in controlling the activity of proteins as well as their function and subcellular localization. To bring MS-based phosphoproteomics to the emerging coral model system Aiptasia, we developed a protocol for phosphosphproteins extraction and generation of a phosphoproteome assay (spectral) library, a comprehensive non-redundant collection of Aiptasia phosphoproteins. Based on the assay library, we then performed accurate label-free quantification (by DIA/SWATH-MS) of the Aiptasia phosphoproteome. Our method reproducibly identifies and quantifies a broad fraction of the phosphorylated proteome of Aiptasia and is comparable to methods developed for other model systems