Proteomics Using Draft Genomes: A Case Study in Spotted Hyena

Crocuta crocuta, the spotted hyena, is unusual among the four remaining hyena species for its adaptation to hunting in preference to scavenging. Individual hyenas alternate between highly social life in matriarchal clans and solo hunting. Until 2020, the molecular biology of this species was hindered by the lack of genome data, but within three years, three draft genomes had been advanced for this species (2020-Yang, 2022-Shao, and 2023-DNA Zoo). This project generated both RNA-Seq (NCBI PRJNA658551) and proteomics data for animals from Kruger National Park in South Africa (ProteomeXchange PXD066654). We evaluated the three draft genomes alongside 2017-SUN, a Trinity de novo assembly of transcript sequences from RNA-Seq data. BUSCO estimated annotation completeness, and Salmon aligned RNA-Seq reads to putative transcript sequences. Proteinortho made it possible to determine which proteins from one database matched protein sequences in another. FragPipe tested the four C. crocuta protein sequence databases in their effectiveness for identifying MS/MS scans in 30 LC-MS/MS experiments, and searches against NCBI Felis catus, Canis lupus familiaris and Mus musculus protein databases evaluated the efficacy of identification against homologous protein sequences. 2020-Yang, the UniProtKB and NCBI reference proteome for C. crocuta, identified only 84.4% as many spectra as did the best performer, 2022-Shao. The F. catus protein database performed almost as well as 2020-Yang, identifying 80.5% as many spectra to orthologous sequences since both species fall within suborder Feliformia. The project enumerated more than 1000 protein sequences that lost multiple peptides when FragPipe used the 2020-Yang database rather than any of the other three sequence databases for C. crocuta. FragPipe identifications from the 2022-Shao assembly show considerable orthology between F. catus organ proteomes and those of the spotted hyena. These proteome identifications provide a first look at differential proteins among lymph nodes from abdominal, head, peripheral, and thoracic regions of the body.