Dataset: Metabolomic data collected during a thermal stress experiment using reef building corals collected in Kāne'ohe Bay, O'ahu, Hawai'i.

From the waters of Kāne‘ohe Bay, HI, four colonies of each coral species M. capitata and P. acuta were identified and collected under SAP 2019-60. Each of the four colonies for each species was fragmented into 30 pieces at the Hawaiʻi Institute of Marine Biology, located on Moku o Loʻe in Kāne‘ohe Bay, HI, and hot-glued to labeled plugs. (21N 157 W ; depth 1m).

Metabolite extraction and analysis performed at Rutgers University.

Metabolite extraction from coral nubbins: Metabolites were extracted using a protocol optimized for water-soluble polar metabolite analysis on LC-MS. The extraction buffer was a solution of 40:40:20 (methanol:acetonitrile:water) (v/v/v) + 0.1 M formic acid. The extraction buffer was stored at −20°C before usage. Immediately preceding the metabolite extraction, 1 ml of extraction buffer was added to a 2-ml glass Dounce homogenizer that had chilled on ice. Pieces of the −80°C preserved nubbins were then clipped, weighed, and added to the cold extraction buffer in the Dounce and left to incubate for 5 min. The pestle of the Dounce was then used to homogenize the coral tissue until there was a visible accumulation of coral skeleton at the bottom of the Dounce and the homogenate was visibly pigmented. An additional 500-μl aliquot of cold 40:40:20 + 0.1M formic acid extraction buffer was then used to rinse down the sides of the Dounce and pestle. The total 1.5-ml volume was then strained through a sterile 100-μm cell strainer into a 50-ml receptacle. There was a visible amount of skeleton collected in the strainer. The rest of the homogenate was then transferred to a 1.5-ml Eppendorf tube, vortexed for 10 s, and then centrifuged for 10 min at 16,000g at 4°C. After centrifugation, there was a pellet at the bottom of the tube. A final 500-μl aliquot of the homogenate was then pipetted to a second clean Eppendorf tube, to which 44 μl of 15% NH4HCO3 was added to neutralize the acid in the buffer. This was the final extract and was ready to be loaded to instrument vials for analysis.

Polar metabolite processing: Polar metabolite extractions were based on Lu et al. (2017). In a glass Dounce homogenizer, samples were mechanically ground in one mL of 40:40:20 (methanol: acetonitrile:water) (v/v/v) + 0.1 M formic acid extraction buffer after incubation in the buffer for 5 min. The sample was transferred to a 1.5-mL Eppendorf tube, with an additional 500 mL of extraction buffer used to rinse the Dounce. The samples were then vortexed for 10 s, before a 10-min centrifugation (16,000g) at 4 C. A total of 500 mL of the homogenate was then transferred to another Eppendorf tube and 44 mL of 15% NH4HCO3 was added to neutralize the extraction buffer. The samples were run on an ultra-high–performance LC-MS (UHPLC-MS), consisting of a Vanquish Horizon UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA) with XBridge BEH Amide column (150 mm by 2.1 mm, 2.5-mm particle size; Waters, Milford, MA, USA), and a Thermo Fisher Scientific Q Exactive Plus with a HESI source.

UHPLC conditions: The HILIC separation was performed on a Vanquish Horizon UHPLC system (Thermo Fisher Scientific, Waltham, MA) with XBridge BEH Amide column (150 mm by 2.1 mm, 2.5-μm particle size; Waters, Milford, MA) using a gradient of solvent A [95%:5% H2O:acetonitrile with 20 mM acetic acid and 40 mM ammonium hydroxide (pH 9.4)] and solvent B [20%:80% H2O:acetonitrile with 20 mM acetic acid and 40 mM ammonium hydroxide (pH 9.4)]. The gradient was 0 min, 100% B; 3 min, 100% B; 3.2 min, 90% B; 6.2 min, 90% B; 6.5 min, 80% B; 10.5 min, 80% B; 10.7 min, 70% B; 13.5 min, 70% B; 13.7 min, 45% B; 16 min, 45% B; 16.5 min, 100% B; and 22 min, 100% B. The flow rate was 300 μl/min. The injection volume was 5 μl, and the column temperature was 25°C. The autosampler temperature was set to 4°C, and the injection volume was 5 μl.

Full-scan MS: The full-scan MS analysis was performed on a Thermo Fisher Scientific Q Exactive Plus with a HESI source, which was set to a spray voltage of −2.7 kV under negative mode and 3.5 kV under positive mode. The sheath, auxiliary, and sweep gas flow rates were 40, 10, and 2 (arbitrary unit), respectively. The capillary temperature was set to 300°C, and aux gas heater was 360°C. The S-lens radio frequency (RF) level was 45. The m/z scan range was set to 72 to 1000 m/z under both positive and negative ionization mode. The automatic gain control (AGC) target was set to 3e6, and the maximum injection time (IT) was 200 ms. The resolution was set to 70,000.

Parallel reaction monitoring MS: The MS2 spectra generation was performed on a Thermo Fisher Scientific Q Exactive Plus with a HESI source, which was set to a spray voltage of −2.7 kV under negative mode and 3.5 kV under positive mode. The sheath, auxiliary, and sweep gas flow rates were 40, 10, and 2 (arbitrary unit), respectively. The capillary temperature was set to 300°C, and aux gas heater was 360°C. The S-lens RF level was 45. The m/z scan ranges were specified for the four dipeptides and monitored for the full 22-min run time. The AGC target was set to 2 × 105, and the maximum IT was 100 ms. The resolution was set to 17,500. The isolation window was set to 2.0 m/z. Collision energy was set to a stepwise 30, 50, and 80 normalized collision energy (NCE). Our results suggest that a single MS2 spectrum may contain irrelevant m/z signals and the pseudospectrum, generated by correlating multiple MS2 spectra to the MS1-extracted ion chromatogram, is an effective approach to “clean” the MS2 spectrum. The pseudospectra were generated using a modified version of COVINA (Su et al. 2020).