Proteomics Using Draft Genomes: A Case Study in Spotted Hyena

Area of Science:

• Genomics and Proteomics
• Comparative Molecular Biology
• Mammalian Evolutionary Biology

Background:

• The spotted hyena (Crocuta crocuta) exhibits unique hunting behaviors, alternating between social clan life and solitary hunting.
• Limited genomic data previously hindered molecular biology studies of C. crocuta.
• Recent advancements include three draft genomes (2020-Yang, 2022-Shao, 2023-DNA Zoo) and generated RNA-Seq and proteomics data.

Purpose of the Study:

• To evaluate the effectiveness of different Crocuta crocuta draft genome assemblies for proteomic identification.
• To compare the proteomic identification capabilities of C. crocuta databases against homologous species.
• To identify differential proteins within C. crocuta lymph nodes.

Main Methods:

• Evaluation of three draft genomes (2020-Yang, 2022-Shao, 2023-DNA Zoo) and a Trinity de novo assembly (2017-SUN) using RNA-Seq data.
• Annotation completeness assessed by BUSCO; RNA-Seq read alignment by Salmon.
• Protein sequence database comparison using Proteinortho and FragPipe for LC-MS/MS data analysis against C. crocuta, Felis catus, Canis lupus familiaris, and Mus musculus databases.

Main Results:

• The 2022-Shao assembly identified 15.6% more spectra than the 2020-Yang reference proteome.
• The Felis catus (domestic cat) protein database showed high orthology, identifying 80.5% as many spectra as the 2020-Yang database, reflecting shared ancestry within Feliformia.
• Over 1000 protein sequences were lost when using the 2020-Yang database compared to others; FragPipe analysis of the 2022-Shao assembly revealed significant orthology with F. catus organ proteomes.

Conclusions:

• The 2022-Shao draft genome assembly is a more effective resource for C. crocuta proteomic studies than the 2020-Yang assembly.
• Comparative proteomic analysis highlights substantial evolutionary conservation between spotted hyena and domestic cat organ proteomes.
• This study provides foundational proteomic data for C. crocuta, including insights into differential protein expression in various lymph node regions.