Distribution of differentially regulated proteins among functional categories. Panel a. vtg1-KO Experiment. Panel b. vtg3-KO Experiment. Only proteins that were identified in > 4 biological samples and that exhibited a > 1.5-fold difference in N-SC between groups (vtg-KO versus Wt), or proteins unique to a certain group, were included in this analysis. In both experiments, the overall distribution of differentially regulated proteins among the functional categories significantly differed between KO and Wt eggs (χ², p < 0.05). Asterisks indicate significant differences between different groups in the proportion of differentially regulated proteins within a functional category (χ², p < 0.05). The corresponding Ensembl Protein IDs and associated gene, transcript and protein names, functional categories (shown above), regulation (compared to Wt) (unique, up-regulated in KO or down-regulated in KO), and fold-difference in N-SC between KO and Wt eggs for proteins included in this analysis for the vtg1-KO and vtg3-KO experiments are given in Tables S1 and S2, respectively

PANTHER GO Biological Processes found to be overrepresented by differentially regulated proteins. Panel a. vtg1-KO Experiment. Panel b. vtg3-KO Experiment. Only proteins that were identified in > 4 biological samples and that exhibited a > 1.5-fold difference in N-SC between groups (vtg1-KO versus Wt), or proteins unique to a certain group, were included in this analysis. Horizontal bars indicate the number of proteins attributed to each GO term for which statistically significant results (Fisher’s Exact test, p < 0.05, followed by Bonferroni correction for multiple testing (p < 0.05)) were observed (Results shown for Wt egg proteins in Fig. 2b. Top Panel based on FDR only, no Bonferroni correction was applied). Numbers next to the bars indicate the fold-enrichment with proteins attributed to each term and the number of asterisks indicates the significance level of the enrichment, as follows p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****). Where possible, horizontal bars are colored to indicate corresponding protein functional categories shown in Fig. 1; cell cycle, division, growth and fate (lavender), protein synthesis (light blue), protein degradation and synthesis inhibition (dark blue), energy metabolism (magenta)

PANTHER GO Molecular Functions found to be overrepresented by differentially regulated proteins. Panel a. vtg1-KO Experiment. Panel b. vtg3-KO Experiment. Only proteins that were identified in > 4 biological samples and that exhibited a > 1.5-fold difference in N-SC between groups (vtg3-KO versus Wt), or proteins unique to a certain group, were included in this analysis. Horizontal bars indicate the number of proteins attributed to each GO term for which statistically significant results (Fisher’s Exact test, p < 0.05, followed by Bonferroni correction for multiple testing (p < 0.05)) were observed. Numbers next to the bars indicate the fold-enrichment with proteins attributed to each term and the number of asterisks indicates the significance level of the enrichment, as follows p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****). Where possible, horizontal bars are colored to indicate corresponding protein functional categories shown in Fig. 1; lipid metabolism (yellow), cell cycle, division, growth and fate (lavender), protein degradation and synthesis inhibition (dark blue)

PANTHER GO Biological Pathways which are significantly over-represented by proteins up-regulated in vtg-KO eggs Panel a. vtg1-KO Experiment Panel b. vtg3-KO Experiment. Only proteins which were identified in > 4 biological samples and those with a > 1.5-fold difference in N-SC, or proteins unique to any group, were included in this analysis. Horizontal bars indicate the number of proteins attributed to each pathway for which statistically significant results (Fisher’s Exact test, p < 0.05, followed by Bonferroni correction for multiple testing (p < 0.05)) were observed. Numbers next to the bars indicate the fold-enrichment with proteins attributed to each pathway and the number of asterisks indicates the significance level of the enrichment, as follows p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****)

STRING Network Analysis of the differentially regulated proteins in vtg1-KO and vtg3-KO experiments. A total of 32 proteins which were down-regulated in vtg1-KO eggs and 94 proteins which were up-regulated in vtg1-KO eggs in vtg1-KO experiment, and a total of 21 proteins which were down-regulated in vtg3-KO eggs and 53 proteins which were up-regulated in vtg3-KO eggs in vtg3-KO experiment, were over-represented in specific biological pathways (Tables 1 and 2). Each network node (sphere) represents all proteins produced by a single, protein-coding gene locus (splice isoforms or post-translational modifications collapsed). Only nodes representing query proteins are shown. Nodes are named for the transcript(s) to which spectra were mapped; for full protein names, see Tables S1 and S2. Edges (colored lines) represent protein-protein associations meant to be specific and meaningful, e.g. proteins jointly contribute to a shared function but do not necessarily physically interact. Model statistics are presented at the top left and at the top right of each panel for proteins down- and up-regulated in KO eggs, respectively. Explanation of edge colors is given below panels. For each experiment, the subnetwork formed by proteins down-regulated in KO eggs is shown to the upper left above the diagonal dashed line, and the subnetwork formed by proteins up-regulated in KO eggs is shown to the lower right below the diagonal dashed line. Where possible, solid lines encircle clusters of transcripts encoding interacting proteins involved in physiological processes distinct from other such clusters (see text for details). Dashed lines identify subclusters of two or more transcripts encoding proteins of a common type

Proteins with significant differences in abundance in vtg1-KO Experiment. Panel a. HeatMap representation of differences in abundance based on normalized spectral counts. Panel b. Vitellogenins with significant differences in abundance between vtg1-KO eggs and Wt eggs. Panel c. Other proteins with significant differences in abundance between vtg1-KO eggs and Wt eggs. All proteins are named for the transcript(s) to which spectra were mapped; for full protein names, see Table S1. Only proteins that were identified in > 4 samples were included in this analysis (t-test, p < 0.05, followed by Benjamini Hochberg correction for multiple tests, p < 0.05). Vertical bars indicate mean N-SC values (N = 4 per group) and vertical brackets indicate SEM. Protein (transcript) labels are color-coded to indicate functional categories to which the proteins were attributed (Fig. 1)

Proteins with significant differences in abundance in the vtg3-KO Experiment. Panel a. HeatMap representation of differences in abundance based on normalized spectral counts. Panel b. Vitellogenins with significant differences in abundance between vtg3-KO eggs and Wt eggs. Panel c. Other proteins with significant differences in abundance between vtg3-KO eggs and Wt eggs. All proteins are named for the transcript(s) to which spectra were mapped; for full protein names, see Table S2. Only proteins that were identified in > 4 samples were included in this analysis (t-test, p < 0.05, followed by Benjamini Hochberg correction for multiple tests, p < 0.05). Vertical bars indicate mean N-SC values (N = 4 per group) and vertical brackets indicate SEM. Protein (transcript) labels are color-coded to indicate functional categories to which the proteins were attributed (Fig. 1)