This post details lipidomics and metabolomics extraction and analysis for samples from the Montipora capitata 2023 larval thermal tolerance project at the Unviersity of Technology Sydney in Australia with Dr. Jennifer Matthews.

See my notebook posts and my GitHub repo for more information on this project.

Briefly, we will be processing larval samples sampled in summer 2023 of Montipora capitata for lipidomics and metabolomics. We will be sampling larvae that were 9 days post fertilization (final time point) incubated at ambient, +3°C, and +6°C for 4 h from wildtype (spawn slick from natural reef), non-bleached (parents that did not bleach under previous bleaching events), and bleached (parents that did bleach under previous bleaching events) parental phenotypes.

How does parental history influence metabolic resilience to thermal stress in symbiotic larvae?

This post contains a log of main activities. All detailed notes and protocols are recorded in my physical lab notebook (Putnam Lab book #31) and backed up with electronic storage in the Putnam Lab Google Drive. Collaborators have asked that the protocols not be publicly posted until published.

Overall, this protocol extracts lipids and metabolites simultaneously, which are then individually obtained by a phase separation step. At the end of the protocol, a protein pellet is used for normalization. This results in lipid, metabolite, and protein quantification for each sample.

April 10, 2024

I arrived at UT Sydney today and stored all samples in the -80°C freezer and delivered fixed larval samples to Jen. Tomorrrow we will begin optimizing protocols.

April 11, 2024

Today, Jen trained me on the lipid and metabolite extraction protocol using test Acropora larvae that she will process for lipid profiles at a later date. We prepared all supplies and reagents that will be used during our work over the next couple of weeks.

We also discussed modifications that will be made to protocols for our Montipora capitata larval samples. The current protocol is for aposymbiotic larvae, and our samples are from symbiotic, vertically transmitting larvae. We are most interested in the host lipid and metabolite profile, because we would like to know how host metabolism and acquisition of translocated products differ depending on parental phenotype and how metabolism and translocation changes under thermal stress.

This therefore requires that we separate the host and symbiont as best as we can to focus on the host response. This will require revisions to the protocol to include host and symbiont fraction separation prior to lipid and metabolite extraction.

Currently, the protocol uses protein quantification at the end of the protocol for normalizing lipid and metabolite profiles to sample total protein. If we perform fraction separations before extraction, we need to quantify protein at the start of the assay.

Therefore, we will optimize the protocol to first remove as much of the symbiont pellet as possible while retaining host lipids followed by protein quantification. We will then use the protein quanitification to proceed with extracting a known amount of tissue protein content as input for lipid and metabolite extractions.

April 12, 2024

Today, we trialed the protocol modifications discussed above. We used 6 samples from the first time point sampling of Hawaii 2023 larval incubations that can be used as trial sampling. We chose these samples to come from different treatment groups, which will leave n=5 samples if we choose to come back to these samples later on. Our experimental samples are all from the final time point sampling. See notebook posts linked at the top of this post for more information on the project.

We first performed host and symbiont fraction separations. We first tried 500uL homogenate volume by removing seawater from the sample by pouring onto a GF/F filter and scraping larvae into a tube with MilliQ water. We then homogenized larvae using a plastic pestle and performed two rounds of centrifuging at 3000 rcf for 5 min. After each round of centrifuging, we removed the supernatant and lipid layers above and around the pellet. This will remove a vast majority of symbiont cells, but will likely not remove all, becuase we need to retain the lipid layer, which sticks to the pellet. This shouldn’t be a problem due to the detection limit likely not being met for symbiont signals due to low densities. We sampled and will look at homogenate for the host fraction to quantify the number of symbiont cells left in the sample. This will balance the need for separation while retaining as much biomass as possible for sampling.

We then quantified protein using a Bradford assay and found that samples were below the detection limit. We then performed another round of separations using a 100uL homogenate volume. We also used 0.22 um Millepore filters and a vacuum pump for removing seawater, which worked better with less larval tissue being stuck to the filter.

We then quantified protein again and found that samples were within detection limits. In our host fraction, we had approx. 50ug of protein in our remaining ~70uL volume. This is lower than Jen prefers for larvae (400ug), but is similar to adult coral values they have used before. If protein values are similar for our experimental samples, we will use the maximum amount possible in the extractions and normalize to the total amount of protein added.

April 14, 2024

Today I labeled tubes for the processing that we will need to do this week. I labeled tubes for host fraction separation (“host” tubes), extraction (“E” tubes), lipid separation (“L”), and metabolite separation (“M tubes”). I also prepared new BSA stocks at 1 mg/mL.

Finally, I processed two samples from the first time point test samples and took subsamples of homogenate of the holobiont and the isolated host fraction. We will use these next week to look at cell densities via microscopy to quantify the presence of cells in host fraction, if any.

April 15, 2024

I separated host fractions for 35 of the 54 our experimental samples today. I followed the protocol we developed on April 12th. The overall process was as follows. All details are in my physical lab notebook.

1. Thaw samples on ice.
2. Add 100 µL LC-grade MilliQ water and 1 µL EDTA (20mg/mL) and 1 µL BHT (20mg/mL) to prevent lipid oxidation. This is our homogenate volume. Chill on ice.
3. Pipette larvae onto a 0.22 µm filter on a vacuum pump. Add another 200 µL of LC-grade water to the original sample tube and pipette onto the filter. This rinses off any remaining seawater and allows for removing all of the larvae from the tube. Use chilled water for all portions of the assay.
4. Scrape the larvae from the filter into a new sample tube with the homogenate water with EDTA and BHT with a chattaway filter. Clean spatula with 80% EtOH between samples.
5. Place on ice.
6. Repeat for all samples.
7. Homogenize all samples on ice with a plastic tube pestel.
8. Centrifuge at 3000 rcf at 4°C for 5 min.
9. Remove the host supernatant and scrape lipid film from around and on top of the pellet. Transfer to a new tube.
10. Centrifuge at 3000 rcf at 4°C for 5 min.
11. Remove the host supernatant and scrape lipid film from around and on top of the pellet. Transfer to a new tube labeled with “Sample - Host”. At this point, the pellet is small or not present.
12. Freeze at -80°C until processing for protein.

This was done for 35 samples (7 batches of 5 samples each). I also generated 3 blanks that will be used during protein quantification that did not have larvae added.

April 16, 2024

Today I finished host separations as described on April 15th.

I then tested protein quantification. We only hve 80-100uL of material after separations and want to retain as much material as possible. So, I tested if a signal could be obtained from just 5uL of sample rather than 10uL in the Bradford assay. It was below the standard curve, so it didn’t work.

Instead of standardizing input protein in our extractions, we will use all available material and standardize to protein after the extraction by using the protein pellet obtained during extractions. I will proceed with extractions starting tomorrow. I’ll start with a batch size of 5 and increase as I become more efficient.

April 17, 2024

I started lipid and metabolite extractions today. This protocol is detailed in my lab notebook. Collaborators asked for the protocol to not be made public until publication, so I am not able to include in my online notebook here.

Today, I ran 4 batches each with 5 samples and 1 blank successfully. I will do another 3-4 batches until finished.

Samples were stored at -80°C for processing next week.

April 18, 2024

I continued with extractions today using the same protocols as yesterday. On the last batch, I was running short on Mix 1 reagents. We do not have the materials to make more. So I calculated the amount needed to finish all samples. In order to do this, I scaled down all reagents and components by 80% and will finish the remaining ~15 samples in two large batches to reduce the number of blanks required. This shouldn’t be a problem. We are adding in such a small amount of material that the extraction will very likely not reach saturation.

April 19, 2024

I finished the last extractions today! All went well. Recorded metadata in notebooks and data files.

April 22, 2024

Today we prepared samples and loaded them on the LCMS. In order to prepare the samples, we dried the lipid extract under a nitrogen stream and then resuspended the extract with a isopropanol and methanol reagent. We added 5 µL of each sample into a pooled tube as a Pooled Biological Quality Control (PBQC). Samples (63 larval samples) were loaded into the LCMS. We are running these cycles with blanks before and after the samples and a PBQC every 9 samples. Samples will be run in negative mode first, then positive mode. This will take about 3 days to run.

April 23, 2024

Today I processed all protein samples using a digestion of the cell debris pellets from extractions. Cell debris was digested with 0.2M NaOH at 98°C for 20 min and then centrifuged with the supernatant used for protein assays.

Assays were run using a Bradford assay. I ran a total of 4 plates. After each plate I calculated the protein concentrations. Any samples that were below the standard curve were run again but with higher input volume. All samples except one worked and were within the standard curve. One sample was below the curve and did not improve with more input material.

LCMS data is looking great. We have data as expected for positive and negative mode with blank samples showing high noise as expected. Samples have clear peaks. This should finish early morning on April 25th.

April 24, 2024

I prepared all metabolite extracts for shipping today to Metabolomics Australia. I generated PBQC samples using a pool of subsamples of all samples. Samples were dried in a vacufuge at 30°C for 3.5 hours and stored in a bag with dessicant. Jen will send these samples on Monday.

We also imaged homogenate samples from two of the first timepoint test samples from the holobiont and host fractions to demonstrate that host tissue did not contain symbionts. We imaged samples on a fluorescent scope and took images showing the presence of host cells (DAPI filter) with symbiont cells present in holobiont fractions and no cells seen in host fraction. Jen will send images to me.

Everything is done! :)