Obtain quote for metabolomics/lipidomics from UW

[AHuffmyer]

Decision that needs to be made at the bottom of this description

Request quote from UW Metabolomics for metabolomic and lipidomic analysis of time series samples:

Samples

Samples:

Species

• A. pulchra
• P. tuahiniensis
• P. evermanni

Sites

• Manava
• Mahana

Time points

• TP1
• TP3
• TP4

Many samples were missing from time point 2 from Acropora (5 out of 10 colonies missing TP2) and Pocillopora (7 out of 10 colonies missing TP2). We do have all time point 2 samples for Porites (0 out of 10 colonies missing TP2).

Decision that needs to be made:

Option 1: Only process TP1, TP3, and TP4 samples for all species = 86 total samples (approx. $22,274).

Option 2: Process TP1, TP3, and TP4 for POC and ACR with all time points for POR = 96 total samples (approx. $24,864).

Are these options in the budget? If so, do we want to go with Option 1 or Option 2? If we have the budget, Option 2 would get us the most data, particularly for Porites.

@sr320 @hputnam do you have a preference for Option 1 or 2?

[sr320]

Option 2

On Wed, Jul 31, 2024 at 4:57 PM Ariana S Huffmyer @.***> wrote:

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[AHuffmyer]

Un targeted metabolite and lipid analyses at UW Northwest Metabolomics:

Assay Name	Assay Description	Platform	Metabolites	Comments
Global LC-MS	Aqueous Global Profiling	Agilent 6546 Q-TOF LC/MS	~800 metabolite IDs + 4000+ unknown metabolites	Aqueous metabolites
	Lipid Global Profiling	Agilent 6546 Q-TOF LC/MS	>1000 lipids & >3000 unknown lipids species	Lipids

Contact: nwmrc@uw.edu

@AHuffmyer submit request for quote

Estimate provided by UW previously including sample prep:

For untargeted and lipid analysis, you would need to provide two separate sample specimens (if possible) since these two assays require totally different sample prep. Our prices per sample are

Quantitative Lipidomics: $99 MS runs + $27 tissue sample prep + $15 BCA protein assay (needed for data normalization) = $141 TOTAL per sample

Global (untargeted) LC-MS profiling: $112 for LC-MS runs + $27 tissue sample prep + $15 BCA protein assay (needed for data normalization) = $154 TOTAL per sample

[hputnam]

@AHuffmyer how much sample do we need compared to what Javi needs for the histone work and compared to the average size of nubbin we have?

[AHuffmyer]

@jarcasariego How much tissue do you require?

I'll inquire with UW for tissue input.

[AHuffmyer]

They recommend 5-10mg of tissue for each assay (will need 10-20 mg tissue for metabolomics and lipidomics). Some of the images of the frozen fragments are very small and I am worried we cannot get that much tissue.

We will need to first homogenize, separate host and symbiont, and lyophilize host fraction. The lyophilized host fraction is what we would submit.

Waiting to get more information from UW facility to ask a few more detailed questions and will chat with Javi about quantity needed for histone work.

If we are limited by tissue, we will need to decide if histone work or metabolomics/lipidomics is higher priority.

[AHuffmyer]

Here is an update on the analysis and information provided from UW:

• They will accept tissue slurry as input rather than dried tissue. The input absolute minimum can be 2mg, but 5-10mg is preferred. This is the weight of tissue that was homogenized, not the weight of the tissue slurry.
• Targeted lipids (~1500 lipids) and targeted metabolomics (~300 metabolites) is the best approach for our work. In this context, targeted means that they only look for known metabolites using in house standards - this gives us plenty of metabolites across lots of pathways. Untargeted will get us unknown metabolites, but we are not going to do compound discovery, so this is not relevant for us. It would produce way too much data to work with and is not what we are interested in.
• Two separate tubes will need to be submitted for each sample (each with the above mentioned tissue input) - one for metabolites and one for lipids, assuming we do both analyses. We can select to just to metabolites if that is all the material we have/budget available.
• Turn around for analysis is about 3 months for full sample set
• We can submit 4 test samples for free. They encouraged this because they want to make sure it works with our given input. These can be run any time and as soon as we like. Turn around is about 2 weeks.
• No quote is needed on their end. They will bill UW budget after sample analysis is completed and data is in hand. We just need to see if approval for our budgets need to be done before analysis on our end.

Quoted sample prices are:

Total price per sample for lipidyzer is $129 (includes MS data acquisition + sample prep + BCA assay for data normalization) and for targeted LC-MS is $134 (includes MS data acquisition + sample prep + BCA assay for data normalization).

Total for both analyses per sample is $263.

[AHuffmyer]

Example data output from the targeted analysis of metabolites and lipids from UW.

2024-06-28_DataSample_Tissue.xlsx 2024-05-20_TargetedData-Tissue.xlsx

[AHuffmyer]

Test samples received at UW, will follow up with group when results are in hand to make decisions on next steps.