# Evolution and adaptations of the seminal proteome in an insect with traumatic insemination ## Description of the data and file structure ### Data archive **File:** `data.zip` This archive contains the data files used in the analyses described in the associated manuscript. The archive contains multiple `.csv`, `.tsv`, and `.rds` files. Missing values are represented either as blank cells or `"NA"`. ------------------------------------------------------------------------ # File descriptions ## Shared reference files The following files are used in multiple analysis scripts. ------------------------------------------------------------------------ ## `DAVID2entrez_geneID.txt` Mapping file used to convert **UniProt protein IDs to Entrez gene IDs** using the DAVID Bioinformatics Resources. Source: https://davidbioinformatics.nih.gov/ **Columns** Column Description ------------- --------------------------- `From` UniProt protein accession `To` Entrez Gene ID `Gene.Name` Gene name annotation **Species:** *Cimex lectularius* ------------------------------------------------------------------------ ## `cimex_signal_peps_combined.rds` List of *Cimex lectularius* proteins predicted to contain **signal peptides**, generated by combining predictions from: - Phobius\ - SignalP 6.0 The file contains UniProt protein IDs predicted to encode secreted proteins. ------------------------------------------------------------------------ ## `chrom_locs.csv` Chromosomal location of each gene inferred from **BLASTn searches against the *Cimex lectularius* chromosome-level genome assembly (Miles et al. 2025)**. **Columns** Column Description ----------- ----------------------------------------- `gene_id` Entrez gene ID `chm` Chromosome on which the gene is located `accession` UniProt ID The *Cimex lectularius* karyotype is: - **Males:** 2n = 26 + X₁X₂Y\ - **Females:** 2n = 26 + X₁X₁X₂X₂ ------------------------------------------------------------------------ ## `codeml_model0_full_results.csv` Evolutionary rate estimates calculated using **PAML codeml (model 0)**. **Columns** Column Description ---------- ------------------------------------------ `GeneID` Entrez gene ID `t` Total branch length across the phylogeny `S` Number of synonymous sites `N` Number of nonsynonymous sites `dN` Nonsynonymous substitution rate `dS` Synonymous substitution rate `omega` dN/dS ratio ------------------------------------------------------------------------ ## `N0.tsv` Hierarchical Orthogroup assignments generated using **OrthoFinder**. Input consisted of protein FASTA files for each species included in the analysis. **Columns** ----------------------------------------------------------------------- Column Description ----------------------------------- ----------------------------------- `HOG` Hierarchical Orthogroup identifier `OG` Orthogroup identifier (deprecated) `Gene Tree Parent Clade` Phylogenetic clade defining the orthogroup ----------------------------------------------------------------------- Species included in the analysis: - Acyrthosiphon\ - Aedes\ - Callosobruchus\ - Chemipterus\ - Cimex\ - Drosophila\ - Gryllus\ - Rhodnius\ - Triatoma ------------------------------------------------------------------------ ## `FlyBaseAllGenes_2_Uniprot_2025_05_07.tsv` Gene ID conversion table downloaded from **FlyBase** for *Drosophila melanogaster*. Source: https://flybase.org/ **Columns** Column Description --------------------- --------------------------- `From` FlyBase gene identifier `Entry` UniProt protein accession `Protein names` Protein name annotation `Gene Names` Gene symbol `Length` Protein length `Gene Ontology IDs` Associated GO terms ------------------------------------------------------------------------ ## `cimex_go_ids.rds` Gene Ontology (GO) annotations for *Cimex lectularius* proteins downloaded from **UniProt**. **Columns** Column Description ------------- -------------------------------------------- `Accession` UniProt protein ID `GOID` Semicolon-separated list of GO identifiers ------------------------------------------------------------------------ # Reproductive biology datasets # `LFQ_v2.0.xlsx` Protein-level quantification and identification results from label-free quantification (LFQ) comparing each tissue/sperm: AGS, Mesa, Sperm, and Testis, each with multiple biological replicates (F1–F17 across 17 fractions/samples). ## Protein Identification & Quality Column Description --------------------------------------------- --------------------------------------------------------------------------------------------------------------------------------------- `Checked` Boolean flag indicating whether the protein entry has been manually selected/verified in PD. `Protein FDR Confidence: Combined` Confidence level assigned based on the combined false discovery rate (FDR) across search engines. Values: High, Medium, Low. `Master` Indicates whether the protein is a Master Protein — the representative entry for a protein group. `Accession` UniProt or database accession number for the identified protein. `Description` Full protein name including organism, taxonomy ID (OX), gene name (GN), protein existence level (PE), and sequence version (SV). `Exp. q-value: Combined` Experimental q-value (FDR threshold) from the combined scoring across search engines; lower = higher confidence. `Sum PEP Score` Summed Posterior Error Probability (PEP) score across all PSMs; higher = better identification confidence. `Coverage [%]` Percentage of the protein sequence covered by identified peptides. `# Peptides` Total number of distinct peptides identified for this protein. `# PSMs` Total number of peptide-spectrum matches assigned to this protein. `# Unique Peptides` Number of peptides that uniquely map to this protein and no other. `# AAs` Length of the protein sequence in amino acids. `MW [kDa]` Theoretical molecular weight in kilodaltons. `calc. pI` Calculated isoelectric point of the protein. `Score Sequest HT: Sequest HT` Total Sequest HT search engine score for this protein. `# Peptides (by Search Engine): Sequest HT` Number of peptides identified specifically by the Sequest HT search engine. `# Razor Peptides` Peptides shared between protein groups but assigned to the highest-scoring protein (razor rule). --- ## Abundance Ratios (Linear Scale) Pairwise ratios of grouped abundance values between the four sample groups. A value >1 indicates higher abundance in the numerator group. Column Description --------------------------------------- --------------------------------------------------- `Abundance Ratio: (Mesa) / (AGS)` Linear fold-change of Mesa relative to AGS. `Abundance Ratio: (Sperm) / (AGS)` Linear fold-change of Sperm relative to AGS. `Abundance Ratio: (Testis) / (AGS)` Linear fold-change of Testis relative to AGS. `Abundance Ratio: (Sperm) / (Mesa)` Linear fold-change of Sperm relative to Mesa. `Abundance Ratio: (Testis) / (Mesa)` Linear fold-change of Testis relative to Mesa. `Abundance Ratio: (Testis) / (Sperm)` Linear fold-change of Testis relative to Sperm. --- ## Abundance Ratios (Log₂ Scale) Log₂-transformed versions of the above ratios. Positive values = upregulated in numerator; negative = downregulated. Column Description --------------------------------------------- --------------------------------------------------- `Abundance Ratio (log2): (Mesa) / (AGS)` Log₂ fold-change of Mesa vs AGS. `Abundance Ratio (log2): (Sperm) / (AGS)` Log₂ fold-change of Sperm vs AGS. `Abundance Ratio (log2): (Testis) / (AGS)` Log₂ fold-change of Testis vs AGS. `Abundance Ratio (log2): (Sperm) / (Mesa)` Log₂ fold-change of Sperm vs Mesa. `Abundance Ratio (log2): (Testis) / (Mesa)` Log₂ fold-change of Testis vs Mesa. `Abundance Ratio (log2): (Testis) / (Sperm)` Log₂ fold-change of Testis vs Sperm. --- ## Statistical Testing of Abundance Ratios Six pairwise comparisons are reported for each statistic: Mesa/AGS, Sperm/AGS, Testis/AGS, Sperm/Mesa, Testis/Mesa, and Testis/Sperm. Column Description ----------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------- `Abundance Ratio P-Value: (X) / (Y)` Raw p-value from the statistical test comparing abundance between the two groups. Blank cells indicate insufficient data for testing. `Abundance Ratio Adj. P-Value: (X) / (Y)` Benjamini-Hochberg adjusted p-value (FDR-corrected) for the pairwise comparison. Used for significance filtering. `Abundance Ratio Variability [%]: (X) / (Y)` Coefficient of variation (%) of the ratio across replicates; reflects measurement consistency between groups. --- ## Grouped Abundances Summarised abundance values per sample group, derived by averaging or summing replicate values after normalisation. Column Description ------------------------------------ ------------------------------------------------------------------------------------ `Abundances (Grouped): AGS` Mean/summed abundance across all AGS replicates. `Abundances (Grouped): Mesa` Mean/summed abundance across all Mesa replicates. `Abundances (Grouped): Sperm` Mean/summed abundance across all Sperm replicates. `Abundances (Grouped): Testis` Mean/summed abundance across all Testis replicates. `Abundances (Grouped) CV [%]: AGS` Coefficient of variation (%) across AGS replicates; reflects within-group reproducibility. `Abundances (Grouped) CV [%]: Mesa` CV across Mesa replicates. `Abundances (Grouped) CV [%]: Sperm` CV across Sperm replicates. `Abundances (Grouped) CV [%]: Testis` CV across Testis replicates. --- ## Per-Sample Raw Abundances (F1–F17) Individual raw abundance values for each sample file before normalisation. Files are grouped by condition: AGS = F1–F5 (5 replicates), Mesa = F6–F9 (4 replicates), Sperm = F10–F13 (4 replicates), Testis = F14–F17 (4 replicates). Column Description ------------------------------------ --------------------------------------------------------------------------------------------------------------- `Abundance: F[n]: Sample, [Group]` Raw MS1 intensity-based abundance value for that specific sample file. Missing values indicate the protein was not quantified in that replicate. --- ## Per-Sample Normalised Abundances (F1–F17) Normalised counterparts of the raw abundance columns, adjusted to correct for sample loading differences or systematic variation between runs. Column Description ------------------------------------------------ --------------------------------------------------------------------------------------------------------------- `Abundances (Normalized): F[n]: Sample, [Group]` Normalised abundance value for each sample/fraction, used for all ratio and statistical calculations. --- ## Detection Status per Sample Boolean/categorical indicator of whether the protein was detected in each individual sample. Column Description ----------------------------------------------- --------------------------------------------------------------------------------------------------------------- `Found in Sample: [Sn] F[n]: Sample, [Group]` Detection status for each sample. Values: **High** (confidently identified), **Peak Found** (signal detected but below high-confidence threshold), or **Not Found** (absent). --- ## Protein Grouping & Modifications Column Description -------------------- --------------------------------------------------------------------------------------------------------------- `# Protein Groups` Number of protein groups this entry belongs to (typically 1 for a unique master protein). `Modifications` Post-translational modifications (PTMs) identified on peptides assigned to this protein (e.g., oxidation, phosphorylation). Blank if none detected. # Sperm-leucylaminopeptidase datasets ## `M17LAP3_SLAP12_GrSm_orthologs.fasta` UniProt protein sequences for S-Lap orthologs and cytosol aminopeptidase (LAP3; P00727) ## `NCBI_BLASTp_SLAPs.csv` BLAST results from NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) giving short name for `first`, `second` and `third` top ranked Drosophila S-Lap for each Cimex `Protein`. Search results were restricted to Drosophila melanogaster ## `reciprocal_best_hits.tsv` Results of local BLASTp for the 13 Cimex S-Lap orthologs against the 6 Drosophila S-Laps. ## `slap_trim.nwk` Newick format tree for Cimex S-Lap orthologs from protein sequences aligned using MAFFT including Dmel and LAP3 then manually pruned to only show Cimex proteins # Protein-ligand complex predictions ## `region_constrained_details.tsv` Results of analysis to characterise M17 aminopeptidase metal-binding and catalytic residue motif of S-Lap and and granny-smith orthologs. ----------------------------------------------------------------------- Column Description ----------------------------------- ----------------------------------- `protein_id` UniProt identifier as provided in the input FASTA file `site_name` Motif site identifier (`metal_1` through `metal_5` = divalent cation binding residues; `cat_1`, `cat_2` = catalytic residues) `position` Position of the best-matching residue in the query sequence (1-based) `residue` Single-letter amino acid code of the residue found at this position `canonical_residues` Residue(s) matching the M17 LAP consensus exactly, separated by `/` `compatible_residues` Residues with structural or mutagenesis evidence for metal coordination in M17 LAPs, separated by `/`; `none` for catalytic sites which have no compatible class `offset` Positional offset of the found residue relative to the exact expected position (0 = exact match; negative = upstream; positive = downstream) `tier` Classification of the found residue: `CONSERVED` = exact match to consensus; `COMPATIBLE` = structurally validated alternative; `NON_FUNCTIONAL` = neither `score` Numeric score for this site: `1.0` = CONSERVED; `0.5` = COMPATIBLE; `0.0` = NON_FUNCTIONAL ----------------------------------------------------------------------- ## AlphaFold3 results Predicted protein--ligand interactions generated using **AlphaFold3 complex structure predictions** (Abramson et al. 2024 Nature). Interactions were predicted between each protein complexed with two zinc ions. Signal peptides were trimmed prior to structure prediction. ### `zn_cifs` Directory containing AlphaFold3 protein--ligand model `mmCIF` files and `key_file.tsv` to match arbitrary file names (e.g., `af_protein_ligand_X_model.cif`) to real UniProt protein ids. ### `zinc_summary_chimerax.csv` Results from measuring zinc ion coordination from `mmCIF` files using ChimeraX v.1.11.1 (Meng et al. 2023; Prot. Sci.). **Columns** ----------------------------------------------------------------------- Column Description ----------------------------------- ----------------------------------- `protein` Protein identifier derived from input CIF filename `zinc_found` Whether zinc ions were detected in the structure (True/False) `zn_index` Index of the zinc ion within the structure (1 = first zinc, 2 = second zinc) `num_zn_total` Total number of zinc ions detected in the structure `site_type` Classified zinc binding site: `Zn1_exchangeable` (lysine- coordinated) or `Zn2_tight` (two aspartate contacts, no lysine) `num_canonical_contacts` Number of canonical M17 coordinating residues after bidentate deduplication `coordinating_residues_raw` All atoms within 3.5 Å of the zinc ion. Format: chain:RES###(atom)=dist Å. Asterisk (*) denotes canonical M17 sidechain contacts `coordinating_residues_deduped` Canonical contacts after bidentate deduplication (closest oxygen retained per Asp/Glu residue). Asterisk (*) denotes canonical M17 sidechain contacts `canonical_residue_types` Residue types of canonical coordinating contacts (LYS, ASP, GLU) `lys_present` Whether lysine coordinates the zinc ion; diagnostic for Zn1 of M17 LAP (True/False) `mean_distance` Mean Zn–ligand distance (Å) across all contacts in the deduplicated set `mean_plddt_at_site` Mean AlphaFold3 pLDDT confidence score across atoms coordinating the zinc ion `score` M17 coordination score (0–10). Incorporates canonical residue presence, coordination number, bond distance quality, and pLDDT confidence `call` Activity classification: `LIKELY_ACTIVE` (score ≥ 6), `UNCERTAIN` (score 3–5), `LIKELY_INACTIVE` (score < 3) `flags` Diagnostic notes from scoring, including missing canonical residues, unexpected coordination numbers, weak or bad distances, low pLDDT, non-canonical contacts, and bidentate deduplication events ----------------------------------------------------------------------- ### `key_file.tsv` Used to match arbitrary filename IDs used in AlphaFold to real protein IDs. Also contains metadata. **Columns** ----------------------------------------------------------------------- Column Description ----------------------------------- ----------------------------------- `filename` Arbitrary ID. Matches `protein` in `zinc_summary_chimerax.csv` `protein_id` UniProt protein ID `num_ligands` Total number of ligands in input .json `ligand_type` Ligand type in input .json `seed1/2/3/4/5` AlphaFold3 model seed for each model ----------------------------------------------------------------------- # Code and software All analysis code is available on GitHub: https://github.com/MartinGarlovsky/Bedbug_proteomics ------------------------------------------------------------------------ # Data access Proteomic data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the identifier PXD075584.