Oct 09, 2019
Metabolite Extraction and Derivatization of Plasma/ Serum Samples for High Resolution GC-MS- based Metabolomics
- Biswapriya Misra1,
- Michael Olivier1
- 1Wake Forest School of Medicine
- Metabolomics Protocols & WorkflowsTech. support email: bbmisraccb@gmail.com
External link: https://onlinelibrary.wiley.com/doi/abs/10.1002/rcm.8197
Protocol Citation: Biswapriya Misra, Michael Olivier 2019. Metabolite Extraction and Derivatization of Plasma/ Serum Samples for High Resolution GC-MS- based Metabolomics. protocols.io https://dx.doi.org/10.17504/protocols.io.723hqgn
Manuscript citation:
Misra, B.B., Bassey, E., Bishop, A.C., Kusel, D.T., Cox, L.A. and Olivier, M., 2018. High‐resolution gas chromatography/mass spectrometry metabolomics of non‐human primate serum. Rapid Communications in Mass Spectrometry, 32(17), pp.1497-1506.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol in our group and it is working. It has been published several times as well.
Created: October 09, 2019
Last Modified: October 09, 2019
Protocol Integer ID: 28475
Keywords: Orbitrap, GC-MS, gas chromatography, mass spectrometry, high resolution, silylation, derivatization, methoxyamine, MSTFA, MeOX,
Abstract
This protocol provides details on preparation of human and non-human primate blood plasma/ serum extraction, and derivatization for GC-MS based metabolomics data acquisition.
Guidelines
1. Avoid freeze thawing of plasma/ serum samples.
2. I prefer to analyze plasma/ serum samples in this order of preference, when available from various source/s investigators: serum (i.e., has no additives)> EDTA-plasma > heparin-Plasma > and NEVER ever the citrate-Plasma (i..e, this citrate can mess up the citrate quantification as well as other TCA cycle metabolites), and never BIO-PAX tub collected materials (i.e, lysis will cause RBC/ WBC contents to contaminate the pure plasma/ serum).
3. No matter what downstream biological question is addressed, prefer to collect and analyze "fasted plasma/ serum" samples to avoid confounders from diet, which has an enormous impact on serum/ plasma metabolites.
4. SUPER IMPORTANT: If you see a red tinge in serum/ plasma avoid it's inclusion in analysis (and inform the source/ PI/ investigator) which means it has lysed blood cell content (i.e., red from hemoglobin) and the measurements would be unreliable and unhelpful altogether.
5. The described protocol for sample preparation and derivatization is a detailed version and adapted from existing literature i.e., :
(a) Lisec, J., Schauer, N., Kopka, J., Willmitzer, L. and Fernie, A.R., 2006. Gas chromatography mass spectrometry–based metabolite profiling in plants. Nature protocols, 1(1), p.387. and
(b) Fiehn, O., 2016. Metabolomics by gas chromatography–mass spectrometry: Combined targeted and untargeted profiling. Current protocols in molecular biology, 114(1), pp.30-4.
while details on the solvent mixtures used for extraction and the extraction steps are available:
(c) Fiehn, O., Wohlgemuth, G., Scholz, M., Kind, T., Lee, D.Y., Lu, Y., Moon, S. and Nikolau, B., 2008. Quality control for plant metabolomics: reporting MSI‐compliant studies. The Plant Journal, 53(4), pp.691-704.
Materials
Acetonitrile, isopropanol, and pyridine, methoxyamine hydrochloride (MeOX), 1% TMCS in N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA), adonitol,
Safety warnings
| Change gloves regularly esp. when suspecting contamination. | |
| Perform all extractions and derivatization in Chemical Hood (not Laminar Flow Hood) | |
| Clean the Chemical Hood work bench with 70% ethanol to get rid of all posisble chemicals/ spills/ DNA/ Protein from previous experiments. | |
| 1.5 mL microcentrifuge tubes need not be autoclaved as long as they are from fresh packets. Use filter tips whenever possible. | |
| Avoid breathing, sneezing, coughing over the samples at any point of time and talk less to avoid salivary droplets/ aerosols to get inside samples. | |
| Get permanent markers, stop watches (timers), Para films on the sides. | |
| Keep all solutions chilled/ on ice always, EXCEPT- Pyridine, MeOX, MSTFA | |
| Dehydrate Pyridine using Sodium Sulfate columns if old/ needed or always keep it sealed under silica- chamber. | |
| Microcentrifuge tubes (1.5 ml, Fisher brand, colorless, low-leaching tubes; protein/ DBA lo-bind does not help, as we are analyzing metabolites and their binding properties ar very different!) should be pre-chilled on ice before transferring solutions. NEVER hold the microcentrifuge tubes with samples inside from bottom with sample in it! Metabolites are susceptible to temperature and thermal degradation | |
| Avoid using perfumes, strong deoderants, medications, flavored food items, confectionaries working in the GC-MS laboratory. | |
| Label all aliquots, tubes, with sample ID and dates all the time. |
Before start
- Before day starts, check the AVAILABILITY of the GC-MS system in first place. As derivatized (methoxyaminated and silylated samples) samples are only stable enough for 24-48 h and their performance decreases there after, better to post-pone sample prpearation than storing the samples in -80 C etc. (not recommended as moisture degrades silylated compounds).
- See that the carrier gasses such as Helium, Nitrogen etc. are at optimal levels in the system.
- Ensure that the GC-MS system is in optimal condition, i.e., perform routine clean-up and maintenance, i.e, LEAK CHECK (using the PFTBA), TUNE, and MASS CALIBRATION on the day of analysis/ prior to the submission que.
- Ensure that the column is in optimal shape by running you favorite RT/ RI calibration standards and blank SOLVENT runs.
- Ensure that new LINER and new SEPTUM are in place. They need to be changed after evry 24 hours of run or 10-20 samples for best reproducible results.
- Prepare fresh stock on MeOX on the day of sample preparation/ derivatization and is stable for a week only.
- Prepare the acquisition que prior to the sample preparation.
- Label the Blank tubes as B1, B2, B3, B4, B5....Bn.
- Label the Reagent Blank tubes as R1, R2, R3,....Rn.
- Label the sample tubes as S1, S2, S3...Sn or with sample codes/ LIMS system generated codes etc.
- Label the QC or pooled QCs as QC1, QC2, QC3, .....QCn etc.
- 10 μl FAME (Supelco, -20 C stored) standards can be used if needed as Retention Time/ Index (LRI) marker or n-hydrocarbon mixtures can be used as Kovat's Index markers.
PROTOCOL OVERVIEW BASED ON Lisec et al., 2006, Fiehn , 2016
PROTOCOL OVERVIEW BASED ON Lisec et al., 2006, Fiehn , 2016
Freshly thawed aliquots of serum or plasma (30 µL) samples from non-human primate (i.e., baboons, vervets) are subjected to sequential solvent extraction once each with 1 mL of acetonitrile: isopropanol: water (3:3:2, v/v ratio) stord in -20 C and 500 µL of acetonitrile: water (1:1, v/v) ratio mixtures stored at 4 °C.22
Adonitol (2 µL from 10 mg/mL stock in methanol, stored in -20 C) is added to each aliquots as internal standard to the sample tube prior to the start of extraction.
The pooled extracts (~ 1500 µL) from the two steps are dried under vacuum at 4 °C prior to chemical derivatization.
Dummy extractions are also performed on blank microcentrifuge tubes that serve as extraction blanks to account for background (extraction solvent, derivatization reagents) noise and other sources of contamination (septa, liner, column, vials, handling etc.).
Blanks are only used to see that no carry overs occurred during randomized run orders and to manually filter out contaminating chemicals from the combined list of features.
The steps involve the addition of 10 μL of MeOX (20 mg/mL stock in pyridine, freshly prepared and stable for a week at maximum) in pyridine incubated under shaking at 55 °C for 60 min followed by trimethylsilylation at 60 °C for 60 min after adding 90 μL MSTFA.
DETAILED PROTOCOL:
DETAILED PROTOCOL:
Prepare methoxyamine (MeOX, Sigma/ Supelco): Prepare a 20 mg/ml MeOX solution in Pyridine; vortex and then Thermomix at 60 °C for 15 min to dissolve; 1 ml is good for a week only if stored in desiccator at room temperature.
3m
Prepare Adonitol ( synonym, ribitol) (Sigma) stock: 2 mg/ ml (Prepare 10 ml, use 20 mg in methanol, i.e., 20 mg/ ml): Store in −20°C for months! Used to (i) monitor extraction efficiency, and (ii) as an internal standard to normalize peak area.
1m
Prepare Extraction Solvent I: 3:3:2 (v/v/v) acetonitrile/isopropanol/water: Make 100 ml (or, as needed): Store in −20°C
1m
Prepare Extraction Solvent II: 1:1 (v/v) acetonitrile/water : Make 50 ml (or, as needed) : Store in -20 °
1m
Retrieve plasma/ serum samples form -80 C freezer on to ice/ dry ice. Can take 30 min to 1 hr for thawing.
Add 2.5µl adonitol (from stock) to each ‘thawed’ sample (i.e., 30 µL plasma/ serum) kept on ice. Hence forth, called "sample".
Add 1 ml cooled extraction solvent to one 1.5 mL microfuge tube containing an aliquot of the sample (30 µl) and ‘dummy’ control (B1, empty tube).
The rest of the tubes should be kept ice chilled (dry ice) at 4°C on ice.
Vigorously vortex for 10 sec and on Thermomixer shake for 5 min at 4°C (1000 rpm shaking needed)
Centrifuge for 2 min at 14,000 × g, room temperature.
Aliquot two (x2) 450 μl portions of the supernatant to separate tubes: one for analysis and one for a backup in -80 C.
Evaporate both aliquots to complete dryness using a speed vacuum concentrator. Preferably operated at 4 C. Store the backup aliquot at −20°C for up to 4 weeks.
Resuspend the other dried aliquot in 450 μL nitrogen-degassed 50:50 (v/v) acetonitrile/water at room temperature.
Centrifuge for 2 min at 14,000 × g, room temperature.
Transfer supernatant (400 μL) to a fresh 1.5-ml polypropylene microcentrifuge tube and evaporate to dryness in a Speedvac evaporator.
Add 10 µl MeOX solution to samples [Stock 20 mg/ ml in Pyridine]. Slow vortex for 10 sec.
1s
Use Thermomixer to heat for 1 hr at 55°C. (no shaking needed)
Add 90 μl MSTFA[ Sigma, Original bottle] to each sample and QC. Slow vortex for 10 sec.
1s
Use Thermomixer to heat for 1 hr at 60°C. (no shaking needed)
Collect the tubes, vortex slowly for for 10 seconds, centrifuge for 30 seconds and collect the supernatant into autosampler vials (with 250 uL inserts, screw capped, Amber color); and submit to the GC-MS autosampler.
2m