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Spotted hyena lymph node, liver, and lung proteomes

ABSTRACT: Proteomics using Draft Genomes: a Case Study in Spotted Hyena Crocuta crocuta, or spotted hyena, are one of four remaining hyena species, notable for its preference for hunting rather than scavenging. Until 2020, no annotated genome had been published for the species; by 2023, three had been published. These bottom-up proteomes, measured in 2020, were used to evaluate the quality of these three genomes for C. crocuta, establishing that the genome annotation featured by UniProt and NCBI lags behind the alternatives in identification sensitivity. Sample handling and mass spectrometry are detailed in 1330MASS_ANALYTICALREPORT.pdf. Notably, these data were produced with Cys alkylated by MMTS rather than iodoacetamide. The search results come from FragPipe 23, using the "Shao.faa" fasta plus contaminants and decoys. These shotgun proteomes were collected in roughly three sets. The three "Test" experiments (March and July 2020) established the amount of peptides loaded on column and asked how peptide diverse the samples were. The 13 RPLC experiments (August 2020) represented peripheral, head, abdominal, or thoracic lymph nodes (PL, HL, AL, or TL) from three different animals (571, 572, and 575) plus a few liver and lung samples. The 12 "Fraction" RAWs (September 2020) represent ten bRPLC fractions of a head lymph node pool. See the QuaMeter.tsv table for more details. These animals are the same as were used for RNA-Seq in NCBI PRJNA658551.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Crocuta Crocuta (ncbitaxon:9678)

SUBMITTER: David L. Tabb

PROVIDER: MSV000098657 | MassIVE | Mon Jul 28 04:56:00 BST 2025

SECONDARY ACCESSION(S): PXD066654

REPOSITORIES: MassIVE

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Project description:Background: RNA-seq based on short reads generated by next generation sequencing technologies has become the main approach to study differential gene expression. Until now the main applications of this technique have been to study the variation of gene expression in a whole organism, tissue or cell type under different conditions or at different developmental stages. However, RNA-seq also has a great potential to be used in evolutionary studies to investigate gene expression divergence in closely related species. Since the more reliable statistical methods for differential gene expression inference are based on the use of raw read count data, the reference genomes of the species to be compared need to be highly comparable. Results: We show that the published genomes and annotations of the three closely related Drosophila species, D. melanogaster, D. simulans and D. mauritiana, have limitations for inter-specific gene expression studies. This is due to missing gene models in at least one of the genome annotations, unclear orthology assignments and significant length differences in the different species. We propose that published reference genomes should be re-annotated before using them as references for RNA-seq experiments to include as many genes as possible and to account for a potential length bias. For that we present a straight-forward reciprocal re-annotation pipeline that allows to reliably compare the expression for nearly all genes annotated in D. melanogaster. We carried out a RNA-seq experiment in combination with quantitative real-time PCR to confirm that the newly generated gene sets do not result in a high number of false positives as observed with references that still show a clear length difference of gene models between species. Conclusions: We conclude that our reciprocal re-annotation of previously published genomes facilitates the analysis of significantly more genes in an inter-specific differential gene expression study. We propose that the established pipeline can easily be applied to re-annotate other genomes of closely related animals and plants to improve comparative expression analyses.

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Spotted hyena lymph node, liver, and lung proteomes

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