NCCOS Assessment: Coral Proteomics on SCTLD Progression in Dry Tortugas across Multiple Species from September 2021-August 2022

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Dry Tortugas

Raw Proteomic Characterization Data from four species of coral collected over the course of a year, during which time, the onset of stony coral tissue loss disease was monitored.

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Project accession: PXD068175

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Individual coral colonies were sampled multiple times over the course of a year (September 2021 - August 2022) to collect samples prior to lesion formation due to disease, and then sampled at the onset of lesion formation. This work was completed by Dr. Grace Klinges (gklinges@asu.edu)

Frozen coral tissue samples were shipped to Dr. Woodley's lab (NOAA) at Hollings Marine Lab (Charleston, SC). The coral tissue was digested for protein isolated and trypsinized peptides were obtained for proteomic characterization.

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Digested peptides were analyzed at University of Arkansas IDEA National Resource for Quantitative Proteomics to characterize the peptides in the coral tissue. The data was run against an Orbicella fasta file and sent back to Dr. Woodley's lab for further analysis

Samples were collected in Dry Tortugas and frozen for molecular analysis

Frozen coral tissue was crushed and placed in lysis buffer (5% SDS with protease inhibitors). 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.

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.

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.

MaxQuant is a quantitative proteomics software package designed for analyzing large mass-spectrometric data sets. It is specifically aimed at high-resolution MS data. Several labeling techniques as well as label-free quantification are supported.