Fish
The samples were collected at the zebrafish facility of the Nord University, Bodo, Norway. The experimental process and husbandry were performed in agreement with the Norwegian Regulation on Animal Experimentation (The Norwegian Animal Protection Act, No. 73 of 20 December 1974). This was certified by the National Animal Research Authority, Norway, General License for Fish Maintenance and Breeding no. 17.
The maintenance of zebrafish was done using an Aquatic Habitats recirculating system (Pentair, Apopka, FL, USA) and following established protocols [31]. The fish were fed newly hatched Artemia sp. nauplii (Pentair) and SDS zebrafish-specific diet (Special Diet Services, Essex, UK) according to the manufacturers’ instruction. The zebrafish used in the experiment were from the AB line.
Sample collection
Embryos originated from natural spawning and were collected at five developmental stages (Fig. 4). Embryo development was monitored and staged according to Kimmel et al. [32]. For each developmental stage, embryo batches were divided into two variants: non-deyolked and deyolked. The non-deyolked (intact) embryos were promptly snap-frozen in liquid nitrogen and subsequently stored at -80˚C. The deyolked embryo variants went through the process of dechorionation (removal of chorion) and deyolking. Additionally, the 1-cell (0.5 hpf) and high-stage (3.3 hpf) embryos were collected to compare our deyolking protocol with that by [16].
Dechorionation and deyolking
Embryos were placed in a Petri dish in phosphate-buffered saline (PBS) supplemented with 1.0 mg/mL Pronase (Sigma Aldrich, St. Louis, MO, USA) [31]. The enzymatic digestion of chorion was performed for 5 min at 37˚C with gentle shaking. Embryos were washed minimum 5 times with PBS or until all visible chorion fragments were removed.
The dechorionated embryos were processed using our modified protocol with three-step deyolking and a single wash. The embryos were transferred to 1.5 mL Eppendorf tubes containing 1.0 mL of deyolking buffer (55 mM NaCl, 3.6 mM KCl, and 1.25 mM NaHCO3) and were mechanically disrupted by pipetting repeatedly through a 100 µL tip. The content was gently mixed by inverting the tube several times before centrifugation at 13,000 RPM for 1 min at 4˚C. The supernatant containing the yolk was discarded, and the pellet was re-suspended with the deyolking buffer, vortexed, and centrifuged as above. The procedure was repeated two times. After this, the pellet was re-suspended with 10 mM Tris-HCl (pH 7.4), vortexed, and centrifuged as above. The supernatant was discarded and the pellet (deyolked embryos) was snap-frozen in liquid nitrogen and stored at -80˚C. Additionally, for comparison of our protocol with that of [16], the dechorionated embryos at 1 cell and high stage were subjected to two types of deyolking protocols reported by [16]: (1) one-step deyolking, and (2) one-step deyolking with two additional wash steps.
Protein extraction
Both intact (non-deyolked) and deyolked embryo samples were lysed by adding 100 µL of sodium dodecyl sulphate (SDS) lysis buffer (1 % SDS; Sigma-Aldrich, St. Louis, MO, USA), 0.5 M triethylammonium bicarbonate buffer pH 8.5 (TEAB; Sigma Aldrich) and 1×Protease Inhibitor cocktail (Thermo Scientific, Rockford, IL, USA)). The tubes were vortexed and incubated at 90˚C for 30 min, then cooled on crushed ice for 5 min. The lysed material was centrifuged at 13,000 RPM for 20 min at 4 ˚C. The supernatant, containing the proteins, was collected and transferred to a new Eppendorf tube. The total protein concentration was quantified using a Qubit® 3.0 Fluorometer (Invitrogen, Eugene, OR, USA) and the Qubit™ Protein Assay Kit (Invitrogen) according to the manufacturer’s instructions. After the quantification, the samples were freeze-dried (VirTis BenchTop™ K, Warminster, USA) at -80 °C for 18 h before being shipped to the Department of Biological Sciences, National University of Singapore for proteomics analysis.
Polyacrylamide gel electrophoresis
One-dimensional gel electrophoresis was performed to check the efficiency of deyolking protocol, as well as to compare the efficiency of our protocol with the previous ones. Approximately equal concentrations of proteins from each sample were supplemented with 2× SDS loading dye. The samples were denatured by incubation at 95˚C for 10 min and then the proteins were separated by SDS gel electrophoresis (4–20% Mini-PROTEAN® TGX™ Precast Protein Gels, Bio-Rad, Hercules, California, USA) in SDS running buffer for 1 h. Afterwards , the gel was washed with deionised water for 10 min. The gel was stained with Coomassie Blue (Coomassie Brilliant Blue R-250, Bio-Rad) for 20 min, and de-stained with de-staining solution (40% methanol + 10% acetic acid) overnight at room temperature.
Tube-gel digestion and sample clean up
For each sample, 30 μg of proteins were used for downstream proteomics analyses. The samples were polymerized in a 10% polyacrylamide gel containing 4% SDS and subsequently fixed with a fixing reagent (50% methanol, 12% acetic acid) for 30 min at room temperature. The gel was cut into small pieces (1 mm3) before being washed three times with 50 mM TEAB/50 % acetonitrile (v/v) and dehydrated with 100% acetonitrile. Next, samples were reduced using 5 mM Tris(2‑carboxyethyl) phosphine (TCEP) at 57 °C for 60 min followed by alkylation with 10 mM methyl methanethiosulfonate (MMTS) for 60 min at room temperature with occasional vortexing. The gel pieces were washed in 500 μL of 50 mM TEAB, dehydrated in 500 μL acetonitrile, and re-hydrated with 500 μL of 50 mM TEAB. The final dehydration step was performed using 100 μL acetonitrile. Trypsinization (1.5 μg trypsin) was performed at 37 °C for 16 h. The digested peptides were centrifuged at 6000 × g for 10 min to collect the supernatant and stored at -20 °C (protocol modified from [17]. The samples were lyophilized and 30 μL of the dissolution buffer (0.5 M TEAB, pH 8.5) was added to each sample.
1D LC-MS/MS analysis
The separation of peptides was performed with an Eksigent nanoLC Ultra and ChiPLC-nanoflex (Eksigent, Dublin, CA, USA) in Trap-Elute configuration. The samples were desalted with a Sep-Pak tC 18 μL Elution Plate (Waters, Miltford, MA, USA), and reconstituted using 20 mL of 2% acetonitrile and 0.05% formic acid. Five mL of each sample was loaded on a 200 μm × 0.5 mm trap column and eluted on a 75 μm × 15 cm analytical column (ChromXP C18-CL, 3 μm). A gradient formed by mobile phase A (2% acetonitrile, 0.1% formic acid) and mobile phase B (98% acetonitrile, 0.1% formic acid) was used to separate the sample content at a 0.3 μL/min flow rate. The following gradient elution was used for peptide separation: 0 to 5% of mobile phase B in 1 min, 5 to 12% of mobile phase B in 15 min, 12 to 30% of mobile phase B in 104 min, 30 to 90% of mobile phase B in 2 min, 90 to 90% in 7 min, 90 to 5% in 3 min and held at 5% of mobile phase B for 13 min (protocol modified from [33].
Protein identification and quantification
Peptide identification was carried out with the ProteinPilot 5.0 software Revision 4769 (AB SCIEX) using the Paragon database search algorithm (5.0.0.0.4767) and the integrated false discovery rate (FDR) analysis function. The data were searched against protein sequence databases downloaded from Uniport on May 2018 (total 119356 entries). The MS/MS spectra obtained were searched using the following user-defined search parameters: Sample Type: Identification; Cysteine Alkylation: MMTS; Digestion: Trypsin; Instrument: TripleTOF5600; Special Factors: None; Species: None; ID Focus: Biological Modification; Database for 2018_May_uniprot-zebrafish.fasta; Search Effort: Thorough; FDR Analysis: Yes. The MS/MS spectra were searched against a decoy database to estimate FDR for peptide identification. The decoy database consisted of reversed protein sequences from the Uniprot zebrafish database. FDR analysis was performed on the dataset and peptides identified with a confidence interval ≥ 95% were taken into account.
KEGG and Gene ontology (GO) functional pathways analysis
To analyse functional pathways associated to protein identified from deyolked and non-deyolked samples, KEGG analysis was performed. The FASTA files were submitted to online server “KAAS - KEGG Automatic Annotation Server” (https://www.genome.jp/kegg/kaas/) in order to get KEGG Orthology (KO) assignments [34]. To map KEGG pathways, the obtained KO numbers were submitted to KEGG mapper web server (http://www.genome.jp/kegg/tool/map_pathway2.html) [35].
GO annotation results and pathway of differentially expressed proteins in pairwise comparisons were obtained using Panther (Panther14.0, 2018_04) ([36]. The web conversion tool (https://biodbnet-abcc.ncifcrf.gov) was used to convert unmapped UniProt Accession IDs to ZFIN ID. The web tool Biomart was used to convert unmapped ZFIN IDs to Gene stable ID and to manually identify the unmapped IDs by gene names [37]. Uniprot was used to identify protein IDs discontinued (deleted) in the 2018_11 release [38].