Abstract
Tetrahymena thermophila is a widely used unicellular eukaryotic model organism in biological research and contains more than 1000 protein kinases and phosphatases with specificity for Ser/Thr/Tyr residues. However, only a few dozen phosphorylation sites in T. thermophila are known, presenting a major obstacle to further understanding of the regulatory roles of reversible phosphorylation in this organism. In this study, we used high-accuracy mass-spectrometry-based proteomics to conduct global and site-specific phosphoproteome profiling of T. thermophila. In total, 1384 phosphopeptides and 2238 phosphorylation sites from 1008 T. thermophila proteins were identified through the combined use of peptide prefractionation, TiO2 enrichment, and two-dimensional LC-MS/MS analysis. The identified phosphoproteins are implicated in the regulation of various biological processes such as transport, gene expression, and mRNA metabolic process. Moreover, integrated analysis of the T. thermophila phosphoproteome and gene network revealed the potential biological functions of many previously unannotated proteins and predicted some putative kinase-substrate pairs. Our data provide the first global survey of phosphorylation in T. thermophila using a phosphoproteomic approach and suggest a wide-ranging regulatory scope of this modification. The provided dataset is a valuable resource for the future understanding of signaling pathways in this important model organism.
Highlights
Tetrahymena thermophila is a species of free-living unicellular ciliated protozoa that is widely distributed in freshwater environments around the world [1]
After all results from three independent experiments had been combined, 1384 phosphopeptides from 1008 phosphoproteins were identified with a false discovery rate of 1.32%, which corresponded to 2238 phosphorylation sites
Details of the identified phosphopeptides, including identifying search algorithm scores and posttranslational modification (PTM) scores, are provided in supplemental Table S1, in which hyperlinks have been included leading to all the mass spectrometry (MS)/MS spectra
Summary
Tetrahymena thermophila is a species of free-living unicellular ciliated protozoa that is widely distributed in freshwater environments around the world [1]. 1384 phosphopeptides and 2238 phosphorylation sites from 1008 T. thermophila proteins were identified.
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