Computational protocol: Ethological principles predict the neuropeptides co-opted to influence parenting

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[…] We converted the resulting RAW spectra using Trans Proteomic Pipeline (Seattle Proteome Center, Seattle, WA, USA). MS/MS spectra were then imported into MASCOT (v2.2.2; MatrixScience, Boston, MA, USA) and searched against all annotated proteins from the published N. vespilloides genome to produce peptide-spectrum-matching scores only. We set search parameters as: enzyme, none; fixed modifications, none; variable modifications as oxidation (M), acetyl (N terminus), pyroglutamic acid (N terminus glutamine), and amidation (C terminus); maximum post-translational modifications, 6; peptide mass tolerance,±1,000 p.p.m.; fragment mass tolerance,±0.6 Da, tolerances set by the machine.We identified proteins with ProteoIQ (v2.6.03; default setting; Premier Biosoft, Palo Alto, CA, USA), which filters and uses MASCOT peptide-spectrum matching scores to statistically validate proteins identifications using the PeptideProphet Protein Probability scoring algorithm. We identified proteins, peptides and assigned spectral counts using all biological replicates within each behavioural state. We only tallied the ‘top-hit' for each spectrum as a further restriction on quantification. We also used ProteoIQ to estimate abundance of neuropeptides after the secondary validation of protein identities. This analysis produces a list of peptides assigned to each identified protein and from this we looked for qualitative differences in the presence/absence of peptides across the behavioural states for peptides that had at least three spectra and were not truncated forms of a larger observed peptide from a particular protein. We excluded peptides from proteins that were only observed in a single behavioural state. We then calculated normalized spectral abundance factor (NASFs) for all proteins within each biological replicate using the protein length for the NASF length correction factor. Only peptides with at least two spectra within one biological replicate were quantified. Neuropeptide proteins were extracted from the overall protein list after establishing their identity within the published N. vespilloides gene set with a Tribolium castaneum neuropeptidome augmented with neuropeptides identified and described from other insects. We confirmed each neuropeptide's identity using NCBI's non-redundant insect protein database. We also assessed whether each neuropeptide had a predicted signal peptide using SignalP (v4.1; ref. ) with a D-cutoff value of 0.34 (ref. ).To test the hypothesis that changes in neuropeptide expression can be predicted a priori, we first performed a multivariate ANOVA to establish that there was an overall difference in the neuropeptide composition between treatments. A significant multivariate analysis allows for univariate a priori contrasts using ANOVA without inflating the Type I error. We followed this multivariate test with univariate tests (ANOVAs) for difference of individual neuropeptide abundance, testing for the effect of behavioural state on abundance. Where the ANOVA was significant, we performed post hoc tests of differences in the pairwise means of the behavioural states using Tukey–Kramer honest significant difference tests, which allow us to compare pairs of behavioural states while controlling for FDR. All statistical analyses were conducted with JMP Pro (v11.0.0, Cary, NC, USA). Raw abundances, the NASF values, of each neuropeptide from every sample were used to calculate composite abundances using principal component analysis. We used R (v3.3.1) using the prcomp function after scaling the raw abundance data for each detected peptide to mean zero and unit variance to calculate PCAs. Visualizations were prepared in R using ggbiplot (v0.90; […]

Pipeline specifications

Software tools ProteoIQ, PeptideProphet, SignalP, JMP Pro
Applications Miscellaneous, MS-based untargeted proteomics
Organisms Nicrophorus vespilloides