Metabolite Extraction Protocol
Rev. 31-Oct-12
Protocol for metabolite extraction of malaria infected erythrocytes with Methanol:water:chloroform (0.6 mL: 0.15 mL: 0.45 mL)
This method will extract both polar and non-polar metabolites, taking advantage of the physico-chemical properties of metabolites by extracting them at three different pHs.
A cell count of control, parasitized (synchronized), and parasitized (unsynchronized) groups will be needed. In addition we will need to measure the total protein content for representative sample from each group.
1. Prepare a circulating water bath containing Ethylene glycol (or equivalent dry ice/isopropanol) at -25°C.
1a. Prepare internal standard stocks in methanol and cool overnight at -20° C (see procedure in step 9 below).
1b. Cool down refrigerated centrifuge/rotor to 4° C.
2. Prepare MilliQ water working stocks, on ice, in 25-50 mL sterile glass vials or glass Erlenmyer flasks of:
(A) ice cold MilliQ water at 1% Formic acid final (i.e. 0.1 mL conc. Formic acid/ 10 mL water), should be pH 2
(B) ice cold MilliQ water, (i.e. ~ pH 7)
(C) ice cold MilliQ water at final of 2% Ammonium Hydroxide, pH 9. This is prepared from vendor stock bottle. Make sure to measure the volume of concentrated ammonium hydroxide carefully in a glass pipette as it drips. Check final pH of working stock waters with pH strips.
3. From the incubator, transfer 0.750 mL erythrocyte aliquots of each erythrocyte group (control or parasitized) of cells into 1.7 mL Microcentrifuge tubes on ice. Spin tubes at 1000g, 2 min., 4° C.
4. Place tube on ice. Aspirate media off erythrocytes (will be variable depending on size of cell pellet). Approximately 0.40-0.45 mL of cell pellet remaining
5. Add 1.5 mL ice cold 1X PBS. Split volume of each tube into three microcentrifuge tubes (~0.5 ml/tube) for processing at three pHs. Spin tubes at 1500g, 1 min, 4 C to pellet cells. Aspirate PBS off erthrocytes (will be variable depending on size of cell pellet). Approximately 0.125 – 0.15 mL of cell pellet will be remaining in each tube.
6. Add 0.15 mL ice cold (MilliQ) water to erythrocytes of either:
(A) ice cold MilliQ water containing 1% Formic acid, pH 2, or
(B) ice cold MilliQ water, ~ pH 7
(C) ice cold MilliQ water containing 2% Ammonium Hydroxide, pH 9. Tap the tubes to resuspend cells.
7. Place tubes in a rack and plunge tubes into the circulating bath at -25° C for 2 minutes seconds, then transfer tubes immediately to a 37° C water bath for 2 minutes
8. Transfer tubes back into a rack at room temperature.
NOTE: We will need confirmation that lysis (and extent, i.e. % of total) has occurred, i.e. pictures of a cell stained control and parasitized (Synchronized and unsynch) at this stage of the protocol. This information will be needed for future manuscript.
9. To each tube add 0.6 mL of -20° C methanol (in glass bottle) containing the diluted (see below) two internal standards: (A) 9-anthracenecarboxylic acid, (B) 1-naphthylamine. Vortex each tube briefly.
· Each Internal Standard vial that was sent to you is dissolved at 10 mg/mL in 100% methanol. Each vial should contain 1.5 mL.
· Dilute each standard at 1:1000 into sufficient volume of methanol (diluted working stock) to last for the whole experiment. Calculate volume of methanol needed for expt. And prepare extra to be safe. (~ if 50 ml needed for this expt. make up 0.050 ml (or 25 ml) of each stock in 50 ml methanol.)
· Prior to use, the methanol internal standard working stock should have been pre-cooled overnight in a -20° C freezer.
10. Add 0.45 mL chloroform to each tube. Vortex each tube vigorously for 30 seconds.
11. Incubate tubes in circulating bath or on dry ice for a total of 30 minutes: Every 5 minutes vortex each tube vigorously for 10 seconds and return to circulating bath.
12. Transfer tubes to rack at room temperature. Add 0.15 mL ice-cold ultra-pure water at desired pH from the Erlenmyer flask stocks to each tube, either 2% ammonium hydroxide in water for pH 9 extracted samples or 1% Formic acid in water for pH 2 tubes. Vortex all tubes and place in microfuge.
13. Spin all the tubes in a microcentrifuge at 1000 g for 1 min at 4° C
14. Check the tubes to see if the phases have separated and that chloroform is at the bottom of the tubes (eyeball it: compare to meniscus on tube). If not separated, add extra 0.1 mL cold MilliQ water (pH adjusted) to each tube—if there is any room left in tube!
15. Transfer all the tubes to -20° C freezer overnight (2-8 h) for any residual chloroform to fall out of aqueous phase into chloroform phase.
16. The next day remove tubes from the freezer. Spin all the tubes in a microcentrifuge at 1000 g for 1 min at 4° C.
17. Assuming the phases have separated clearly, transfer exactly 0.6 ml (or as much as you can get) of the top phase (methanol/water aqueous phase) to a fresh tube. Seal tubes with parafilm before shipping. (The following steps will be done by Theo: Add an equivalent volume of acetonitrile (ie. to give a 1:1 ratio) Vortex and transfer to 4° C (or on ice) and incubate for 20 minutes.
18. Spin all the top phase tubes in a microcentrifuge at 13,000 g for 10 min at 4° C
19. A pellet might be visible at the bottom of the tubes. Be careful to avoid it and transfer the supernatant to fresh tubes. Then discard tube with pellet. Make sure tubes are not threaded. For shipping: Use parafilm to wrap tightly around the tops of the tubes. Place in a -20° C freezer until ready to ship.
For immediate analysis by LC/MS do the following:
· Dry down (No heat) the Methanol/water/acetonitrile in a SpeedVac as this solution cannot be filtered using the 10 kDa Pall ultracentrifuge filter tubes
· Resuspend dried residue at bottom of tube in 0.5 mL of 1:1 methanol/MilliQ water (< 18 ohm)
· Transfer 0.5 mL to 10kDa ultrafiltration membrane tube and centrifuge at 3000g for 1 minute (or until filtered completely)
· Dry the filtrate in a SpeedVac (as before with no heat)
· Resuspend dried tube in 100 uL of 1:1 methanol/water + 0.1% formic acid
· Inject between 1-5 uL into LC/MS; adjust final inj. volume based on analysis of number of saturated features
· Store tube of remaining solvent (100- x uL) at -20° C
20. Chloroform phase: Transfer the chloroform phase (below the erythrocyte disc) to fresh tubes, being careful to avoid disturbing the disc.
21. Spin all the bottom phase (chloroform) tubes in a microcentrifuge at 13,000 g for 1 min at 4° C. Transfer chloroform to fresh tubes, being careful to avoid any pellet at bottom of the tube. Dry down the chloroform phase. Store at -20° C freezer until ready to ship. Alternatively, store at -80° C for long term.
21. When ready to analyze chloroform phase, resuspend in 100 uL methanol/0.1% formic acid. Inject 1-5 uL into LC/MS
21. Alternatively, if samples are not going to be used, place the samples in a box and use tape or some other means to secure the tubes in the box so that they don’t move around.
* Send samples on plenty of dry ice. Request shipping to ship box upright so that tubes are not upside down or on their side.
LC-microTOFQ conditions
Antimalaria lc-qtof method with diamond hydride column
A 15mM ammonium acetate in water
B 15mM ammonium acetate in acetonitrile
Flow rate: 0.2ml/L
Runtime: 35min
Column temperature: 45 0C
Gradient program:
0-5min 100% B; 5-26min 100%B to 100%A; 26.01-35min 100%B
tofq: ESI, - (negative)
capillary, 3500V; Nebulizer, 2 bar; dry gas, 8 l/min; dry temp, 195; funnel 1 RF, 300Vpp; funnel 2 RF, 300Vpp; Hexapole RF, 250 Vpp; collision energy, 10 eV; collision RF, 200 Vpp; transfer time 80 us; pre puls storage 8.0 us; rolling average 3x; spectra rate 1 Hz.
Mass range, 50-1000 m/z
A 0.2% acetic acid in water
B 0.2% acetic acid in acetonitrile
Flow rate: 0.2ml/L
Runtime: 35min
Column temperature: 450C
Gradient program:
0-5min 100% B; 5-26min 100%B to 100%A; 26.01-35min 100%B
ESI, + (positive) capillary, 4500V; Nebulizer, 2 bar; dry gas, 8 l/min; dry temp, 195; funnel 1 RF, 300Vpp; funnel 2 RF, 300Vpp; Hexapole RF, 200 Vpp; collision energy, 10 eV; collision RF, 100 Vpp; transfer time 80 us; pre puls storage 10 us. rolling average 3x; spectra rate 1 Hz.
Mass range, 50-1100 m/z