# Proteogenomic Reassessment Provides Novel Insight into the Life Cycle of Tetrahymenathermophila

## TL;DR

This study improves the gene catalog of Tetrahymena thermophila and reveals new insights into its life cycle regulation using proteogenomics.

## Contribution

A novel proteogenomic approach validated thousands of genes and identified new genes and post-translational modifications in Tetrahymena thermophila.

## Key findings

- 24,319 predicted genes were validated, and 383 novel genes were identified.
- 7123 post-translational modification sites were mapped across 4705 proteins.
- The study provides a comprehensive resource for understanding life cycle regulation in Tetrahymena thermophila.

## Abstract

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 10 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 7123 modification sites distributed across 4705 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.

•Proteogenomics integrates T2T genome, transcriptome, and proteome of T. thermophila.•Protein expression mapped across 10 distinct life cycle states.•24,319 genes validated, 383 novel genes and 7123 PTM sites identified.•Dynamics of protein expression and PTMs underpin life cycle regulation studies.•Valuable resource for ciliate biology and cross-species comparative research.

Proteogenomics integrates T2T genome, transcriptome, and proteome of T. thermophila.

Protein expression mapped across 10 distinct life cycle states.

24,319 genes validated, 383 novel genes and 7123 PTM sites identified.

Dynamics of protein expression and PTMs underpin life cycle regulation studies.

Valuable resource for ciliate biology and cross-species comparative research.

This study presents a comprehensive proteogenomic analysis of Tetrahymena thermophila across 10 life cycle states. By integrating high-resolution mass spectrometry with a telomere-to-telomere genome assembly, we validated over 24,319 predicted genes and discovered 383 novel genes. Additionally, we mapped 7123 post-translational modification sites, revealing dynamic regulatory mechanisms linked to development and environmental response. This work provides a valuable resource for advancing functional genomics and understanding molecular regulation in this key model organism.