Data Management Plan (Deprecated)

This dataset is comprised of proteomics results to better understand gamete compatibility factors. The dataset includes gametes from three species, Acropora cervicornis, Acropora Palmata, Orbicella faveolate, which were collected in three locations near Key Largo, Florida (Ofav: Horseshoe Reef, Apal: North Dry Rocks, Acer: Carysfort) during three different time periods (August 2022, August 2023, and September 2023).

Reef Location Bounding Box where corals were originally sourced.

Lineage Statement:
These data were generated after collecting gametes spawned from Acropora palmata, Acorpora cervicornis, and Orbicella faveolata off the coast of Key Largo in August 2022 and August/September 2023.

Process Steps:

• Fertilization trials were conducted by performing reciprocal crosses. Gamete bundles from a single coral genet were collected in a 15 mL tube and incubated at room temperature until bundles lyse. Sperm were transferred to a clean 15 mL tube without disrupting ova. Sperm were diluted and concentration was calculated using a cellometer. Sperm were diluted and added to the appropriate vials. Ova were washed with artificial sea water and added to the appropriate vials. The gametes were incubated together for 4-6 hours prior to fixing for counting. (Citation: Collection of gametes in Key Largo 2022 and 2023)
• After gametes were combined for fertilization assays, the remaining sperm and ova were preserved for other molecular analyses. For glycan imaging, sperm and ova were fixed and stored at 4*C until processing. (Citation: Collection of gametes in Key Largo 2022 and 2023)
• Gametes from collected bundles were frozen at -20*C in protein lysis buffer (5% SDS with protease inhibitor). The proteins were extracted by tip sonicating on ice and then centrifuged at 2,000 rcf for 5 minutes. The supernatant was transferred to a new tube and then centrifuged at 10,000 rcf for 10 minutes. The supernatant was transferred to a new tube and 50 ul was reserved for protein quantification. Protein was quantified using Pierce BCA Protein Assay Kit. (Citation: Collection of gametes in Key Largo 2022 and 2023)
• Based on quantification, a volume equivalent to 100 ug was diluted to a volume of 80 ul in 5% SDS. Samples were reduced with 10 ul DTT at 60*C for 10 minutes. After cooling to room temperature (10 minutes), samples were alkylated with calcium acetamide and incubating in dark for 30 minutes. Alkylation was quenched by adding 12% phosphoric acid. Protein samples were digested following Protofi's S-trap protocol, using a 1:20 trypsin:sample ratio and incubating for 2 hours at 47*C. The resulting peptides were evaporated until dry and resuspended in 100 ul 0.1% formic acid in water. Peptide concentration was quantified using Pierce Quantitative Fluorometric Peptide Assay kit. (Citation: Collection of gametes in Key Largo 2022 and 2023)
• Peptides in solution were sent to University of Arkansas and analyzed using LC-MS/MS analysis. Peptide and proteins were identified using Mascot running against a known Orbicella faveolata annotated metagenome. Resulting search data was imported to Scaffold and identifications were accepted at <1% local FDR for peptides and proteins. Normalized spectral abundance factor was derived from normalized spectral counts and intensity based on absolute quantification (iBAQ) intensity data. (Citation: Collection of gametes in Key Largo 2022 and 2023)

Proteomic Analysis

Peptides digested from isolated proteins were identified from annotated genomes

Project Name: Proteomics of gametes collected from Acropora cervicornis, Acropora palmata, Orbicella faveolata

Project accession: PXD057823

Token: DJjiY09bxcWh

Project DOI: Not applicable

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