Multi-step Preparation Technique to Recover Multiple Metabolite Compound Classes for In-depth and Informative Metabolomic Analysis

The overall goal of this procedure is to demonstrate an effective means of fractionating metabolites in complex biological fluids in order to obtain simplified samples for improved coverage of the metabolome. This is accomplished by first spiking each sample with internal standards to monitor the reproducibility of sample preparation and instrument conditions. The second step is to add ice cold methanol to each sample to precipitate the proteins which may otherwise interfere with subsequent chromatography and analysis by mass spectrometry.

Next, the liquid liquid extraction step separates the hydrophilic from the hydrophobic metabolites. The final step is solid phase extraction to further fractionate the lipid metabolites into phospholipids fatty acids and neutral lipids. Ultimately, this combined multi-step method is used to show effective separation, excellent reproducibility, and improved coverage of the plasma metabolome.

The main advantage of this technique over existing methods, such as a simple methanol extraction, is that we obtain improved metabolite coverage, especially of lipids, which is important when performing metabolomics profiling studies or when lipids are of particular interest. Prior to protein precipitation, prepare the samples first thaw samples to room temperature. Next, spike the samples with internal standards ISTD that were prepared beforehand.

All standard concentrations have been adjusted as necessary based on the sensitivity of the MS and HPLC instrumentation used. For analysis, spike each sample with 10 microliters. Each of hydrophilic and hydrophobic standard solutions spike each sample with 10 microliters of either one x two x or four x positive control solution.

After all samples have been spiked with internal standards and positive control solutions, vortex each sample for 10 seconds. To begin protein precipitation, add 400 microliters of ice cold methanol to each sample. Vortex for 10 seconds per tube centrifuge at zero degrees Celsius for 15 minutes at 18, 000 times.

G, A protein pellet should form on the bottom of the tube. After centrifugation transfer all the supernatant to a new glass culture tube and then dry under nitrogen. This dried residue will undergo liquid liquid extraction later.

For analysis of the protein pellet fraction, add one milliliter of methyl turt beetle ether or MTBE to the white or off-white protein pellet vortex for 30 seconds per tube and centrifuge at zero degrees Celsius for 15 minutes at 18, 000 times. G decant the MTBE layer to a new glass culture tube. Since the size of the pellet will vary among samples, it is important to consistently aspirate the same amount of MTBE for all samples.

For example, if only 900 microliters can be decanted for the sample with the least amount of supernatant, then decant 900 microliters. For all samples, Add another one milliliter of MTBE to the protein pellet vortex for 30 seconds, and then centrifuge at zero degrees Celsius or 15 minutes. Add 18, 000 times G as before aspirate the MTBE layer and add to the glass culture tubes prepared earlier.

Dry the samples by nitrogen flow and resuspend in 200 microliters of one-to-one chloroform methanol. Transfer each sample to a centrifuge tube and centrifuge at zero degrees Celsius for 15 minutes at 18, 000 times G.After that, use glass pipettes to transfer the supernatant to autos sampler screw cap vials. To begin this procedure, use a glass pipette to add three milliliters of MTBE to the dried methanol residue prepared earlier and vortex for 30 seconds.

After that, add 750 microliters of water to each tube and vortex for 10 seconds. Centrifuge at about 200 times G for 10 minutes at room temperature, two distinct layers are visible After centrifugation, carefully aspirate 2.5 milliliters of the top MTBE layer without pipetting the aqueous layer below and transfer to a clean glass culture.Two. Add three milliliters of MTBE to the remaining water part of each sample.

And vortex 10 seconds per tube centrifuge at about 200 times G for 10 minutes. At room temperature, aspirate three milliliters of MTBE with without getting water and combine with the previous MTBE tube. This MTBE fraction will undergo solid phase extraction Later.

Concentrate the remaining aqueous layer by drying under nitrogen. Resuspend the residue in 100 microliters of one, and add 400 microliters of ice cold methanol to each tube vortex, and then transfer to a micro centrifuge tube. Leave at negative 80 degrees Celsius for 20 to 30 minutes to allow any remaining protein to precipitate out in the methanol.

Next centrifuge at zero degrees Celsius for 15 minutes at 18, 000 times G, there should be a precipitate along the side of the tube after centrifugation aspirate 450 microliters of supernatant without aspirating the precipitate and transfer to a clean micro centrifuge tube. Dry completely in a vacuum centrifugal concentrator at no more than 45 degrees Celsius for about one to two hours. Resuspend the dried supernatant in 200 microliters of 5%acetyl nitrile water and vortex.

Briefly transfer samples into autos, sampler, vials, and freeze at negative 80 degrees Celsius. To begin the procedure for solid phase extraction, dry the MTBE fraction obtained previously under a good flow of nitrogen at 35 to degrees Celsius for 10 to 15 minutes. When the MTBE fractions are completely dry, stop the flow of nitrogen and quickly resuspend each sample in one milliliter of chloroform using a glass pipette vortex.

Briefly wash and condition the solid phase extraction or SPE cartridge twice with 400 microliters of hexane. Discard the waste and replace with a new glass collection tube. Add the sample to the SPE column, apply vacuum and collect the flow through.

With a glass pipette, add one milliliter of two to one chloroform isopropyl alcohol to the SPE column and collect the flow through in the same glass tubes. This is the neutral fraction. Dry the neutral fraction under nitrogen for 10 to 15 minutes.

To minimize oxidation with a glass pipette, add one milliliter of 5%acetic acid in ethyl ether to the SPE column and collect the flow through. This is the fatty acid fraction. Dry the fatty acid fraction under nitrogen for 10 to 15 minutes to minimize oxidation using plastic tips.

Add 800 microliters of methanol to the SPE cartridge and collect the flow through In 15 milliliter plastic conical tubes, this is the phospholipid fraction. Transfer the phospholipid fraction to 1.5 milliliters centrifuge tubes, dry the samples with a vacuum centrifugal concentrator at 45 degrees Celsius for about one to 1.5 hours. Lastly, reus suspend each of the samples from the three fractions in 200 microliters of 100%methanol transfer to autos, sampler, vials, and store in the negative 80 degrees Celsius freezer.

The effectiveness of the M-T-B-E-S-P-E method in extracting both lipid standards and endogenous metabolites is demonstrated overall better extraction and coverage of the metabolites were obtained compared to other methods such as methanol extraction or MTBE only extraction when the number of features was compared using qualitative and quantitative software. Following lc MS analysis, the comparison of M-T-B-E-S-P-E fractions showed minimal overlap among the three fractions following SPE, which demonstrates the efficiency in separating the hydrophobic metabolites into their respective chemical classes for more confident metabolite identification. In addition, the recovery of the internal standards in fractions using the M-T-B-E-S-P-E method, NR 12 demonstrated that the internal standards were alluded in the fraction related to their chemical class.

To evaluate chromatographic reproducibility of the data sample preparation was performed on three separate pooled plasma QC samples, and each sample was injected in triplicate on the LCM MSS instrument. The consistent overlap demonstrates instrument and sample preparation reproducibility. The increase in chemical noise observed for the negative ionization mode of the fatty acid fraction may be due to contaminants in the LCM S solvents.

Therefore, only metabolites that alluded prior to nine minutes were analyzed. Don't forget that working with chloroform, MTBE, ethyl ether, and even the more common reagents used in this method can be extremely hazardous and precautions such as wearing a lab coat, gloves, and eye protection and performing sample preparation in a fume hood should always be taken while performing this procedure.