Sample fractionation prior to LC-MS/MS in the egg quality experiments.

<p><b>Panel a</b>. Sample fractionation after SDS-PAGE in the Pooled Samples Experiment (N = 4 spawns per egg quality pool). <b>Panel b</b>. Sample fractionation after SDS-PAGE in the Multiple Samples Experiment (N = 4 replicate spawns per egg quality type). For each experiment, numbered frames in red indicate fractions excised from the gel and processed separately before submission to LC-MS/MS. Intact lipovitellin (Lv) heavy chain (closed arrows) and light chain (open arrows) were excluded in the Multiple Samples Experiment to better resolve other proteins. In total, 56 egg protein fractions were individually subjected to LC-MS/MS and downstream proteomic analyses. HMW; High molecular weight, LMW; Low molecular weight.</p

Enrichment analysis of differentially expressed proteins overexpressed in specific biological pathways.

<p>Enrichment analysis of differentially expressed proteins overexpressed in specific biological pathways.</p

Network of differentially expressed proteins overexpressed in specific biological pathways.

<p>Shown are the results of <i>Search Tool for the Retrieval of Interacting Genes/Proteins</i> (STRING) network analysis of the 74 non-redundant proteins that were differentially regulated in good versus poor quality eggs and were over-represented in specific biological pathways in the PANTHER Pathways enrichment analyses (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188084#pone.0188084.s003" target="_blank">S3 Table</a>). Proteins up-regulated in good quality eggs are shown to the right of the solid grey line and proteins up-regulated in poor quality eggs are shown to the left of the line. Each network node (sphere) represents all proteins produced by a single, protein-coding gene locus (splice isoforms and post-translational modifications collapsed). Only nodes representing query proteins are shown. Nodes are named for the transcript(s) to which spectra were mapped, with the text colored according to the processes shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188084#pone.0188084.g002" target="_blank">Fig 2</a>; for full protein names, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188084#pone.0188084.s001" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188084#pone.0188084.s002" target="_blank">S2</a> Tables. Small nodes represent proteins of unknown 3D structure. Large nodes represent proteins for which some 3D structure is known or predicted. Edges (lines) represent protein-protein associations meant to be specific and meaningful, i.e. proteins jointly contribute to a shared function but do not necessarily physically interact. Model statistics are presented at the lower left. Explanation of edge colors is given on the lower right. Dashed lines encircle groups of transcripts involved in the named biochemical pathways or physiological processes. See text for details.</p

Enrichment of biological process gene ontology (GO) terms with proteins up-regulated in the Multiple Samples Experiment.

<p>Shown are the results of PANTHER over-representation tests for enrichment of Biological Process GO terms with proteins up-regulated in good and poor quality zebrafish eggs in the Multiple Samples Experiment. <b>Top Panel</b>. Good quality eggs. <b>Bottom Panel</b>. Poor quality eggs. Horizontal bars indicate the number of proteins attributed to each GO term for which statistically significant results (χ², <i>p</i><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 <i>p</i>≤0.05 (*), <i>p</i>≤0.01 (**), <i>p</i>≤0.001 (***), and <i>p</i>≤0.0001 (****). Where possible, horizontal bars are colored to indicate corresponding protein functional categories shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188084#pone.0188084.g002" target="_blank">Fig 2</a>; energy metabolism (magenta), protein synthesis (light blue), cell cycle, division, growth and fate (lavender), endosome/lysosome-related activities (brown). For poor quality eggs, GO terms preceded by an asterisk involve cytoskeletal activities. See text for details.</p

Proteins highly up-regulated in good or poor quality eggs in the Pooled Samples Experiment.

<p>The proteins are named for the transcript(s) to which spectra were mapped; for full protein names see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188084#pone.0188084.s001" target="_blank">S1 Table</a>. These proteins were either unique to an egg quality type or they showed a ≥5-fold difference in N-SC between egg quality types, placing them in the top 2.3% of proteins showing such differences. <b>Left Panel</b>. Proteins highly up-regulated in good quality eggs. <b>Right Panel</b>. Proteins highly up-regulated in poor quality eggs. Where possible, protein (transcript) labels are color-coded to indicate functional categories to which the proteins were attributed (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188084#pone.0188084.g002" target="_blank">Fig 2</a>). Arrows indicate proteins unique to an egg quality type. Asterisks indicate proteins detected and regulated in the same direction in the Multiple Samples Experiment.</p

Proteins significantly differing in abundance between egg quality types in the Multiple Samples Experiment.

<p>The proteins are named for the transcript(s) to which spectra were mapped; for full protein names, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188084#pone.0188084.s002" target="_blank">S2 Table</a>. Only proteins common to both egg quality groups with ≥4-fold difference in N-SC between groups, placing them in the top 2.3% of proteins showing such differences, or proteins unique to an egg quality group with mean N-SC values ≥0.5 and a statistically significant difference in N-SC between egg quality groups, are shown (independent samples t-test, <i>p</i>≤0.05 after Benjamini Hochberg correction for multiple tests). Vertical bars indicate mean N-SC values (n = 4 per egg quality type) and vertical brackets indicate SEM. Protein (transcript) labels are color-coded to indicate functional categories to which the proteins were attributed (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188084#pone.0188084.g002" target="_blank">Fig 2</a>). Asterisks indicate proteins that were also detected and regulated in the same direction in the Pooled Samples Experiment.</p