Guide for metabolomics experiments

This guide provides a good practical foundation to guide our users through basic steps involved in metabolomic analysis by Mass spectrometry. These guidelines can be applied to different types of samples to improvement the quality of the results.

You MUST completely AVOID!!

STABILIZERS: DO NOT USE any glycerol, PEG or related stabilizers. They are easy to ionize, especially PEG, and if they are present, that is all you will see!

COLORED TUBES AND TIPS: DO NTO USE IN ANY STEP.  These tubes often contain residual quantities of dyes that can be release into your sample. We provide some options "mass spec friendly" list.

DO NOT SEAL TUBES WITH PARAFILM. They are a rich source of PEG contamination.

A COUPLE OF VERY IMPORTANT THINGS TO AVOID OTHER CONTAMINANTS

1. Any sample manipulation should be done in a BSC, laminar flow hood, or by using mask and disposable surgical cap.
2. Always wear nitrile (not latex) gloves.
3. Wear a lab coat and make sure there is no gap between your coat sleeve and the gloves (lab tape and rubber bands work well).
4. All bottles and other containers used should be purchased for and dedicated to MS metabolomics work. Do not wash with detergent. Do not share with others in your lab for non-MS purposes.

 RECOMMENDED REAGENTS AND MATERIALS

SOLUTIONS

i  All solutions must be fresh prepared

i  Make enough solution for all sample

i  All reagents must be at least HPLC grade. For more information consult the list of recommended material.

Extraction solution A = 100% Methanol containing one IS for positive mode and one for negative mode.

Extraction solution B = 0.1% Formic Acid in Waters (LC-MS grade) containing one IS for positive mode and one for negative mode.

Extraction solution C = Methanol/Water (1:1) containing one IS for positive mode and one for negative mode.

Extraction solution D = Dichloromethane/methanol (3:1) containing one IS for positive mode and one for negative mode.

Extraction solution E = Chloroform/Methanol/Water (1:3:1 ratio) containing one IS for positive mode and one for negative mode.

Extraction solution E = 80% Methanol containing one IS for positive mode and one for negative mode.

Note: Make sure the drug has not been used by subjects.

i  Important information

Study design

Definition of technical and biological replicates depends on the model.

Quality Control (QC)

The purpose of QCs is to monitor the performance of metabolomics workflows (extraction and analysis)

„ Prepare QC sample(s) by mixing equal volumes from each of the samples in the batch. The ideal scenario is do it before extraction.

Preparation of the QCs should follow the same procedure performed for samples including number of freeze-thaw cycles.

If it is not possible to create a pooled QC sample due to limited sample amounts commercially available QC sample may be used (ex: human serum)

Internal standard(s)  Added in first step of sample extraction, generally at the extraction solution.

Randomize  Randomization of sample preparation order to avoid systematic bias

Chapter I – Sample Label

-       All samples MUST to be labelled or bar-coded and an Excel sheet with provided before MS analysis.

-       Label must be made on both, lids and sides of the tubes, with water resistant markers or bar code tags.

-       The labelling system should be unique and contain PI initials (two letters), in charge student initials (two letters) and numbered form 001-1000… (figure 1).

-       All the tubes MUST to be in a Microtube Storage Boxes

-       All Storage boxes MUST have a tag in the lid (Figure 2) containing all important information about samples.

Ex: PI: Robert Powers, Student: John Doe, Samples 1-10

                                Label as F RPJD001, RPJD002 ….RPJD010

NOTE: IT IS ESSENTAIL THAT WE RECEIVE A SPREADSHEET DETAILING ALL SAMPLES THAT WILL BE SENT TO OUR LABORATORY. This is to guarantee we have organized appropriate storage facility well in advance of receiving samples.

Chapter II – Sample collection, storage and transport

1. 1.       BODY FLUIDS

i Please BE CAREFULLY when handling samples affected by disease.

i Take care not to generate aerosols and ALWAYS wear lab coat, mask, googles and gloves.

1.1.       Blood, Serum and Plasma

1.1.1 BLOOD COLLECTION

-   Devices used for blood collection are always a source of contaminants. We suggest purchase enough quantities of these important consumables to last for the duration of the study.

- The choice of anticoagulant for metabolomics applications is still a subject of discussion and there is a lack of clear recommendations. Based on current evidences we recommended EDTA (ethylenediaminetetraacetic acid) or Heparin as anticoagulant over sodium citrate.

- Fill 3 device with ultrapure water and note as blank1-3. This will be treated same way as your sample.

-   Avoid hemolyzed samples by placing whole blood immediately in ice cold water after collection.

- Keep samples on ice (or at around 4 ˚C) until finish all sample collection.

-   Transfer 500 µL aliquots of blood (make at least 2 from each subject in order to have a backup sample for any potential repeat analysis) in to new 2 mL labelled tubes (DO NOT USE COLLORED TUBES) using a pipette with appropriate filter tip

- Stored the samples at -80 °C until analysis and, if needed, all transportation should be carried out under dry ice. PLEASE FOLLOW CHAPTER I RULES FOR LABELING.

1.1.2 PLASMA OBTENTION

- After blood collection as described before, transfer samples to refrigerated centrifuge and spin the blood samples for 15 min at 2000 g at 4 °C to move the blood cells to the bottom of the tube.

- Open the tube carefully to avoid disturbing the pellet or causing splashing of the blood.

- Immediately transfer 500 µL aliquots of plasma (make at least 2 from each subject in order to have a backup sample for any potential repeat analysis) in to new 2 mL labelled tubes (DO NOT USE COLLORED TUBES) using a pipette with appropriate filter tip (check options at the material list).

- Stored the samples at -80 °C until analysis and, if needed, all transportation should be carried out under dry ice. PLEASE FOLLOW CHAPTER I RULES FOR LABELING.

1.1.3 SERUM OBTENTIOM

i  Devices used for blood collection are always a source of contaminants. We suggest purchase enough quantities of these important consumables to last for the duration of the study.

- Fill 3 device with ultrapure water and note as blank1-3. This will be treated same way as your sample.

- After blood collection let the tube sit upright at room temperature for a minimum of 30 min to a maximum of 60 min to allow clot formation.

- After clot formation, centrifuge samples at 1500 × g at 4 °C for 20 min.

- Immediately transfer 500 µL aliquots of serum (make at least 2 from each subject in order to have a backup sample for any potential repeat analysis) in to new 2 mL labelled tubes (DO NOT USE COLLORED TUBES) using a pipette with appropriate filter tip (check options at the material list).

- Stored the samples at -80 °C until analysis and, if needed, all transportation should be carried out under dry ice. PLEASE FOLLOW CHAPTER I RULES FOR LABELING.

 1.2.             Urine

1.2.1 HUMAN SAMPLE COLLECTED AT HOME

- Provides 1 container for collection (container A) and 3 containers (B, C and D) for aliquots to the subject.

- Give a written orientation to subject explain how to collect urine according to clinical protocol previously defined (as timed or 24 h collection).

- Instruct the patient collect the urine into container A and immediately aliquoted into containers B, C and D and freeze immediately after aliquot has made.

- All transportation should be carried out under dry ice. If dry ice is not possible, samples MUST BE transported in ice.

- Upon arrival at the laboratory, stored the samples at -80 °C until analysis. PLEASE FOLLOW CHAPTER I RULES FOR LABELING.bNOTE: KEEP the same numbers of thaw cycles for all sample.

1.2.2 HUMAN SAMPLE COLLECTED AT LABORATORY

- Provides a suitable and well labeled container for the subject.

- After collection make aliquots in 2 mL tubes previously labeled.

- Stored the samples at -80 °C until analysis and, if needed, all transportation should be carried out under dry ice. PLEASE FOLLOW CHAPTER I RULES FOR LABELING.

1.2.3 RODENT SAMPLE

i  Animals should be keep it individually in metabolic cages during the whole experiment duration. This is important to minimize stress caused by environmental change.

- Before collection cleaned the bed with ultrapure waters from all cages.

- Place a container, containing 0.5% of sodium azide, immersed in dry ice and let the urine been collected for the time previously defined.

- After collection make aliquots in 2 mL tubes previously labeled.

- Stored the samples at -80 °C until analysis and, if needed, all transportation should be carried out under dry ice. PLEASE FOLLOW CHAPTER I RULES FOR LABELING.

 1.2.       ANIMAL AND HUMAN TISSUES

i  Please BE CAREFULLY when handling samples affected by disease.

i  Take care not to generate aerosols and ALWAYS wear lab coat, mask, googles and gloves.

i  Perform all homogenization and extraction operations in an appropriate fume cupboard;

- The timing of tissue collection for control and treatment groups should be randomized

- If rodents are the subject do not use anesthetic, perform the sacrifice by cervical dislocation.

- Tissue collection should be performed as fast as possible.

-  After collected, rinse tissues with ice cold PBS, blotted with lint free tissue paper.

- If possible, cut the tissue into 2 or 3 pieces and aliquot (in order to minimize freeze-thaw cycles) and immediately snap-frozen in LN₂.

- Stored the samples at -80 °C until analysis and, if needed, all transportation should be carried out under dry ice. PLEASE FOLLOW CHAPTER I RULES FOR LABELING.

 1.3.       ADHERENT AND SUSPENSION CELLS

i  This is a general procedure and the results can vary according to cell type. In general, a range such as 5×10⁶ up to 1×10⁷ cells (Note: cells/cm² for adherent and cells/mL for suspension cells) will be suitable, but this is solely dependent on the cell type in question. It is always imperative to run small trial to optimize for your case.

2.1 Adherent Cells

-       For control purposes, please make blank medium which must be incubated but without cells.

-       Carefully remove all supernatant from cells (keep an aliquot back for medium analysis if desired).

-       Wash cells quickly with blood bank saline 0.9% at 37 °C (with enough volume to fully cover the cellular monolayer) at room temperature. Note: Do not use PBS. Unbuffered saline helps remove residual media, waste products, cell debris, and phosphate salts. Do not use pure water once cause osmotic shock and can rupture cells.

-       Remove all blood bank saline 0.9% by pipetting carefully until that no solution remains. Note: If your cells are weakly adherent is imperial to have extra care to not disturb the cells, as this will result in biomass loss.

-       To quench the cells, add 1 mL of pre-chilled (−50 °C) 60% aqueous methanol supplemented with 70 mM HEPES agitate the flask to ensure that all cells are covered. Note: the volume should be previously optimized and enough to fully cover the cellular monolayer.

-       Use a disposable cell scraper to remove all the cellular monolayer from the flask surface.

-       Harvest the quenching solution/cell mixture using a disposable pipette and transfer to a labelled Eppendorf or Falcon tube (depending on volume) previously labeled. PLEASE FOLLOW CHAPTER I RULES FOR LABELING.

-       Procedure to extraction step or snap frozen in LN₂ and stored at −80 °C until analysis and, if needed, all transportation should be carried out under dry ice.

2.2 Suspension Cells

-       Gently mix the cell suspension.

-       Remove an aliquot of cell suspension from the flask and count the cells using an appropriate cell counting method.

-       Transfer the volume containing the desired cell number to a new labeled 2.0 mL tube containing 80 mg zirconium beads. PLEASE FOLLOW CHAPTER I RULES FOR LABELING. 

-       Centrifuge the cells at low speed (5 min, 500-800 g, room temperature) to gently pellet the cells.

-       Aspirate and discard the culture medium (keep an aliquot back for medium analysis if desired).

-       Immediately and carefully add 1.8 mL warm blood bank saline 0.9% (37 °C). Note: Do not use PBS. Unbuffered saline helps remove residual media, waste products, cell debris, and phosphate salts. Do not use pure water once cause osmotic shock and can rupture cells.

-        Shake a little bit. DO NOT VORTEX.

-       Remove and discard all solution.

-       To quench the cells, add 1 mL of pre-chilled (−50 °C) 60% aqueous methanol supplemented with 70 mM HEPES and vortex to ensure that all cells are covered.

-       Procedure to extraction step or snap frozen in LN₂ and stored at −80 °C until analysis and, if needed, all transportation should be carried out under dry ice.

1. 4.       CELL CULTURE MEDIUM 

This is a general procedure and the results can vary according to cell type. In general, a range such as 50 to 1000 µL will be suitable, but this is solely dependent on the cell type in question. It is always imperative to run small trial to optimize for your case.

-       Remove cell medium from either adherent or suspension pellet and transfer to a new labelled tube. DO NOT USE COLOR TUBES.

-        centrifuge samples at 13,500 g for 5 min AT 4 °C.

-       Transfer the supernatant for new labelled tube. Make aliquots (≈200 µL) to always have a backup. PLEASE FOLLOW CHAPTER I RULES FOR LABELING.

-       Procedure to extraction step or snap frozen in LN₂ and store the clarified medium at –80 °C until analysis and, if needed, all transportation should be carried out under dry ice.

Note: If both cell and media analysis are desired first pellet the cells at low speed and then proceed to high speed centrifugation step.

1. 5.       MICROBIAL CELLS 

i  This is a general procedure and the results can vary according to cell type. We suggest run a small trial to determine how much culture media volume with (1, 5, 10, 20 & 50 mL) is necessary to get enough biomass to generate a suitable instrument response. PLEASE FOLLOW CHAPTER I RULES FOR LABELING.

-       Measure the optical density (OD₆₀₀) for each culture in solution (required for LC-MS normalization).

-       Inactivate the cells metabolism by cooling down the samples on ice. Please note that alternative quenching strategies can be used.

-       Once cooled down, centrifuge samples at 4800 g for 10 min at 4 °C to pellet cellular mass.

-       If desirable, save the medium for analysis (see Cell culture medium procedure)

-       Wash the cell pellet with 1X phosphate buffered saline (PBS) with enough volume to cover the pellet.

-       Discard the PBS and centrifuge samples at 4800 g for 2 min at 4 °C and remove residual by pipetting.  

-       Procedure to extraction step or snap frozen in LN₂ and stored at −80 °C until analysis and, if needed, all transportation should be carried out under dry ice.

Chapter III – Metabolites extraction

This is a general procedure and the results can vary according to sample. You can use as a start point to optimize for you experiment.

i Please BE CAREFULLY when handling samples affected by disease.

i Take care to not generate aerosols and ALWAYS wear lab coat, mask, googles and gloves.

i FOLLOW CHAPTER I RULES FOR LABELING TUBE

i DO NOT thaw more than 24 samples at any single point. You may not be able to manage more, and this could compromise the final analysis.

i ALWAYS run a “Process blank”. To do this, perform all the extraction steps applying the same solvents, chemicals, consumables, and standard operating procedure, as for the test samples, but in the absence of any actual biological sample.

1. 1.       Serum/Plasma

-       Prepare and separates all material and solutions before you start. Check recommended material list.

-       Allow serum/plasma samples to thaw on ice at 4 °C for 30–60 min.

-       While samples are thawing, prepare one tube labeled as QC (pooled quality control). NOTE: In this tube You will put together one aliquot of all you sample (we recommend 10% of the volume used in extraction). For that, make sure to pick up a tube able to fit the final volume capacity. Ex: For extraction 0f 24 plasma sample you need a tube able to fit 12 ml, 9.6 of extraction solution and 2.4 mL of sample (pool of 10 µL from each sample).

-       Vortex and transfer 100 µL of each sample to a new 2 mL containing 400 µL ice cold extraction solution A.

-       Transfer 10 µL of each sample to QC tube containing ice cold extraction solution A (Keep 1:4 vol/vol).

-       Vortex for 15 s and pellet the protein precipitate in a centrifuge operating 4 °C and at 15,800 g for 15 min.

-       Transfer supernatant to a new labeled 2.0-ml tubes (please check chapter I) and freeze-drying samples in a centrifugal vacuum.

„ PAUSE POINT Store the samples at -20 °C for up to 12 weeks.

1. 2.       Urine

-       Prepare and separates all material and solutions before you start. Check recommended material list.

-       Allow the samples thaw in ice.

-       While samples are thawing, prepare one tube labeled as QC (pooled quality control). NOTE: In this tube You will put together one aliquot of all you sample (we recommend 10% of the initial sample volume). For that, make sure to pick up a tube able to fit the final volume capacity.

-       Centrifuge samples at 10,000 g for 10 min at 4 °C to remove particulates.

-       Transfer 50 μL to a labeled Maximum recovery vials containing 100 μL extraction solution B and mix well.

-       For QC sample, transfer 5 μL of each sample for the QC tube.

-       Add extraction solution B (1:2 sample vol:Sol B vol) and vortex. Ex. For 30 urine samples, the final volume it will be 150 μL. So, the volume of sol B it will be 300 μL.

-       PAUSE POINT Store the samples at -20 °C or below for up to 12 weeks.

1. 3.       Animal and Human tissues

3.1.  Sample homogenization

-       Prepare and separates all material and solutions before you start. Check recommended material list.

-       Allow the samples thaw in ice.

-       While samples are thawing, prepare two tubes labeled as QC (pooled quality control). NOTE: One tube will be for polar (QCp) fraction and the other for non-polar fraction (QCnp).

-       Pulverize the frozen tissue using a cryomill (recommended) or LN₂precooled mortar and pestle

-       Lyophilize pulverized overnight.

3.2. Metabolites extraction  

-       Weigh 20 mg from the homogenized dried tissue powder was weighed (25 mg) into a precooled 2mL cryotube.

-       Prepare two tubes labeled as QC (pooled quality control). NOTE: One tube will be for polar (QCp) fraction and the other for non-polar fraction (QCnp). If only one LC method is used you may combine the fractions and in this make only one QC tube.

-       Add 700 μL of ice-cold extraction solution C (Polar extraction).

-       Add 80 mg of zirconium beads and beats for 10 s and cool down on ice for 30 s. Repeat the cycle 3 more times.

-       Centrifuge samples at 10,000 g for 10 min at 4 °C.

-       Collect 80 μL the supernatant and transfer to the QCp tube.

-       Collect the remaining supernatant it to a new labeled tube (polar fraction). NOTE: make aliquots of 200 µL.

-       Place the tubes containing fraction polar in dry ice.

-       IMMEDIATELY after collection of polar fraction, add 700 μL of ice-cold extraction solution D (Non-polar extraction).

-       Repeat the homogenization cycle and centrifuge as previous described.

-       Collect 80 μL the supernatant and transfer to the QCnp tube.

-       Collect the supernatant and transfer it to a new labeled glass tube (non-polar fraction). NOTE: make aliquots of 200 µL.

-       Freeze-drying polar fraction in a centrifugal vacuum.

-       Freeze-drying the non-polar extract in the glass cupboard (centrifugal vacuum IS NOT compatible with chloroform).

-       „ PAUSE POINT Store the samples at -80 °C for up to 12 weeks.

1. 4.       Adherent and suspension cells

-       Prepare and separates all material and solutions before you start. Check recommended material list.

-       Allow the cells thaw in ice.

-       Prepare two tubes labeled as QC (pooled quality control). NOTE: One tube will be for polar (QCp) fraction and the other for non-polar fraction (QCnp). If only one LC method is used, you may combine the fractions and in this make only one QC tube.

-       Add 200 µL of ice-cold extraction solution A to the samples.

-       Homogenize cells by beating for 10 s and cool down on ice for 30 s. Repeat the cycle 3 more times.

-       Centrifuge samples at 10,000 g for 10 min at 4 °C.

-       Collect the supernatant into a new labeled tube (polar fraction).

-       Keep the tubes in dry-ice and procced for next step.

-       IMMEDIATELY after collection of polar fraction, add 1 mL of ice-cold extraction solution E (Non-polar extraction).

-       Repeat the homogenization step.

-       Centrifuge the mixture at 15000 g and 4 °C for 10 min.

-       From the bi-phasic system created, transfer the upper layer to the tubes containing polar fraction.

-       Carefully, aspirate the bottom organic layer and transfer into another 2 mL Eppendorf tube.

-       From polar fractions, take 10% of each sample and transfer to the QCp. Do the same procedure for non-polar fraction (QCnp tube).

-       Freeze-drying polar fraction in a centrifugal vacuum and the non-polar extract in the glass cupboard (centrifugal vacuum IS NOT compatible with chloroform).

-       „ PAUSE POINT Store the samples at -80 °C for up to 12 weeks.

1. 5.       Microbial cells

-       Prepare and separates all material and solutions before you start. Check recommended material list.

-       Prepare two tubes labeled as QC (pooled quality control). NOTE: One tube will be for polar (QCp) fraction and the other for non-polar fraction (QCnp). If only one LC method is used, you may combine the fractions and in this make only one QC tube.

-       Allow the cells thaw in ice.

-       Add 1 mL of ice-cold extraction solution F to the cells pellets.

-       Vortex until pellet dissolves.

-       Transfer each sample to a new pre-cooled 2 mL tube containing 80 mg zirconium beads.

-       Homogenize cells by beating for 10 s and cool down on ice for 30 s. Repeat the cycle 3 more times.

-       Centrifuge the solution at 10,500 g for 10 min at 4 °C.

-       Collect the supernatant into a new 2 mL labeled tube (polar fraction).

-       Keep the tubes in dry-ice and procced for next step.

-       IMMEDIATELY after collection of polar fraction, add 1 mL of ice-cold extraction solution E (Non-polar extraction).

-       Repeat the homogenization step.

-       Centrifuge the mixture for 10 min at 15000 g and 4 °C.

-       From the bi-phasic system created, transfer the upper layer to the tubes containing polar fraction.

-       Carefully, aspirate the bottom organic layer and transfer into another 2 mL Eppendorf tube.

-       From polar fractions, take 10% of each sample and transfer to the QCp. Do the same procedure for non-polar fraction (QCnp tube).

-       Freeze-drying polar fraction in a centrifugal vacuum and the non-polar extract in the glass cupboard (centrifugal vacuum IS NOT compatible with chloroform).

-       PAUSE POINT Store the samples at -80 °C for up to 12 weeks.

References:

Tuck et al. J Proteome Res. 2009 Jan; 8(1): 113–117.

Yin et al. Anal Bioanal Chem. 2015; 407(17): 4879–4892.

Dunn et al. Nature Protocols. 2011; 6: 1060–1083

Want et al. Nature Protocols. 2010 Jun;5(6):1005-18

Masson et al. Anal. Chem. 82, 7779–7786 (2010).

Want et al. Nature Protocols. 2012 Jun;5(6):1005-18

Muschet et al. Metabolomics (2016) 12: 151.