Proteogenomic Reassessment Provides Novel Insight into the Life Cycle of Tetrahymena thermophila.

Tetrahymena thermophila (T. thermophila), a well-established model organism, has been instrumental in advancing our understanding of evolutionarily conserved biological processes. A key biological feature of this unicellular eukaryote is its life cycle strategy, marked by three major stages: growth, starvation and conjugation. Despite its prominence as a model system, functional genomic studies of T. thermophila have been constrained by limitations in the accuracy and completeness of gene discovery since the initial genome assembly in 2006. To address this gap, we performed a multi-stage proteogenomic analysis, combining genomic sequencing with high-resolution mass spectrometry (MS)-based proteomic profiling across ten strategically selected life cycle states. This integrative approach enabled a comprehensive reassessment of gene discovery, leading to the validation of 24,319 previously predicted protein-coding genes and the identification of 383 novel genes. Additionally, our investigation systematically identified a diverse repertoire of post-translational modifications (PTMs), including 7,123 modification sites distributed across 4,705 proteins. These PTMs are postulated to exert critical regulatory functions during developmental phase transitions. Collectively, this work not only refines the T. thermophila gene catalog and enhances its utility as a robust genetic toolkit for advancing biological research but also offers new mechanistic insights into the molecular regulation of its life cycle progression. Keywords: Tetrahymena thermophila; proteogenomics; mass spectrometry; genome assessment; post-translational modification.