Overview
In this video, we demonstrate SPME-probe-based extraction of metabolites from a pre-harvested donor kidney. The extracted metabolites can be used to perform metabolomic and lipidomic analysis in order to assess the organ quality for transplantation.
Protocol
All procedures involving human participants have been performed in compliance with the institutional, national and international guidelines for human welfare and have been reviewed by the local institutional review board.
1. Preparation of Probes
- Prepare titanium-nickel alloy probes (40 mm length; 0.2 mm diameter) coated with 7 mm mix-mode sorbent. The number of probes depends on the time points targeted during the entire procedure and the number of replicates (3 is recommended per time point).
NOTE: The length and type of extraction phase may be adjusted based on the mode of study, the polarity of metabolites, and the sample matrix. - Prepare a cleaning mixture composed of 2:1 chloroform:methanol (v/v). Pipette 1.0 mL of the solution to each 2.0 mL glass vial and place one probe, previously pierced through the cap, in each vial.
NOTE: Before use, clean all probes to remove contaminating particles. - Put the vials on the agitator and set the agitation speed at 1,200 rpm. After 45 min, stop the device and rinse the coatings with LC-MS grade water.
- As coatings must undergo a preconditioning step to activate them, prepare a preconditioning mixture composed of 1:1 methanol:water (v/v). Pipette 1.0 mL of the solution to each 2.0 mL glass vial and place one probe, previously pierced through the cap, in each vial.
- Put the vials on the vortex agitator and set the agitation speed at 1,200 rpm.
- After 60 min, stop the agitator and rinse the coatings with LC-MS grade water.
- Sterilize probes according to the standard surgical equipment sterilization protocol.
2. Extraction
- Open the sterile package right before sampling to ensure a sterile environment.
- Insert two probes directly into the kidney cortex for 10 min at each time point. The entire length of the coating must be covered by the tissue matrix; no specific angle is required, but ca. 90 deg is usually used.
NOTE: The entire medical procedure follows standard protocols in a given institution. No modification concerning SPME sampling is considered. The procedure involves the six following sampling time points: a) before kidney resection, in vivo from donor; b)–e) after 1 h, 3 h, 5 h, 7 h of kidney perfusion, ex vivo in organ chamber; f) after reperfusion, in vivo from recipient. - Retract the probes by pulling them out from the tissue and then rinse coatings with LC-MS grade water to clear any remaining blood from the coating surface. Rinse away from the surgical site, and immediately after removal of the probes.
3. Transport and Storage
- Place probes in separate vials and close them.
- Place vials in a Styrofoam box filled with dry ice or in liquid nitrogen for transport.
- Store samples in a freezer (-80 °C), or immediately commence the desorption step.
4. Desorption
- Prepare desorption solutions composed of 80:20 acetonitrile:water (v/v) for metabolomic analysis, and 1:1 isopropanol:methanol (v/v) for lipidomic analysis.
- Pipette 100 μL of the solution to inserts placed into 2.0 mL labeled vials and place one probe, previously pierced through the cap, in each vial.
- Put the vials on the vortex agitator and set the agitation speed at 1,200 rpm for 120 min.
- Remove probes from the vials. Obtained extracts are now ready for instrumental analysis.
5. LC-MS analysis
- Place vials containing extracts in the autosampler of the LC-MS system.
NOTE: Liquid chromatography (RP, HILIC) coupled with high-resolution mass spectrometry and an orbitrap mass analyzer were used for this study. For metabolomic analysis parameters, go to step 5.2. For lipidomic analysis parameters, go to step 5.6. - Use a pentafluorophenyl (PFP) column (2.1 mm x 100 mm, 3 μm) for reversed-phase separation.
- Set the flow rate to 300 µL/min, and autosampler and column temperatures to 4 °C and 25 °C, respectively.
- Prepare mobile phases according to the following proportions: mobile phase A: water:formic acid (99.9:0.1, v/v), and mobile phase B: acetonitrile:formic acid (99.9:0.1, v/v). Set the mobile phase flow according to the following parameters: starting mobile phase flow (0–3 min): 100% A followed by a linear gradient to 10% A (3–25 min), ending with the isocratic flow of 10% A until 34 min, followed by 6 min of column re-equilibrium time.
- For HILIC separation, use a HILIC column (2.0 mm x 100 mm, 3 μm, 200A). Set the flow rate to 400 μL/min.
- Prepare mobile phases according to the following proportions: mobile phase A: acetonitrile:ammonium acetate buffer (9:1, v/v, effective salt concentration 20 mM), mobile phase B: acetonitrile:ammonium acetate buffer (1:1, v/v, effective salt concentration 20 mM).
- Set the mobile phase flow according to the following parameters: start mobile phase flow (0–3 min) at 100% A, hold for 3.0 min and then ramp to 100% B within 5 min. Hold with 100% B until 12 min, followed by 8 min of column re-equilibrium time.
- Set the scan range to 85–1000 m/z. Set HESI ion source parameters in positive ionization mode to: spray voltage 1,500 V, capillary temperature 300 °C, sheath gas 40 a.u., aux gas flow rate 15 a.u., probe heater temperature 300 °C, S-Lens RF level 55%.
- Run QC samples composed of 10 µL of each analyzed sample (regularly, every 10–12 samples) to monitor instrument performance.
- For reversed-phase separation, use a C18 column (2.1 mm x 75 mm, 3.5 μm).
- Set the flow rate to 200 µL/min, and autosampler and column temperatures to 4 °C and 55 °C, respectively. Prepare mobile phases according to the following proportions: mobile phase A: H2O:MeOH (60: 40, v/v), 10 mM ammonium acetate and 1 mM acetic acid; mobile phase B: IPA:MeOH (90:10, v/v), 10 mM ammonium acetate and 1 mM acetic acid.
- Set the mobile phase flow according to the following parameters: 0–1 min (20% B), 1–1.5 min (20–50% B), 1.5–7.5 min (50–70% B), 7.5–13 min (70–95% B), 13–17 min (95% B), 17–17.1 min (95–20% B), 17.1–23 min (20%).
- For HILIC separation, use a HILIC column (100 x 2.1 mm, 3 μm).
- Set the flow rate to 400 µL/min, and autosampler and column temperatures to 4 °C and 40 °C, respectively.
- Prepare mobile phases according to the following proportions: mobile phase A:ACN; mobile phase B: 5 mM ammonium acetate in water. Set the mobile phase flow according to the following parameters: 0–2 min (96% B), 2–15 min (96–80% B), 15–15.1 min (80–96% B), 15.1–21 min (96% B).
- Set HESI ion source parameters in positive ionization mode to spray voltage 3,500 V, capillary temperature 275 °C, sheath gas 20 a.u., aux gas flow rate 10 a.u., probe heater temperature 300 °C, S-Lens RF level 55%.
- Run QC samples composed of 10 µL of each analyzed sample (every 10–12 samples) to monitor instrument performance.
- Perform data acquisition in software compatible with the instrument.
6. Data Analysis
- Perform data processing, putative identification, and statistical analysis using software dedicated to untargeted metabolomics and lipidomics analysis.
NOTE: Principal components analysis (PCA) and box-whisker plots can be obtained to visualize data structure.
Subscription Required. Please recommend JoVE to your librarian.
Materials
Name | Company | Catalog Number | Comments |
Acetic acid | Merck | 5330010050 | Mobile phase additive |
Acetonitrile | Alchem | 696-34967-4X2.5L | HPLC solvent |
Ammonium acetate | Merck | 5330040050 | Mobile phase additive |
BENCHMIXER XL MULTI-TUBE VORTEXER | Benchmark Scientific | BV1010 | Vortex mixer |
Caps | Perlan Technologies | 5183-2076 | Blue scrw tp, pre-slit PTFE/Si spta, 100PK |
Chloroform | Merck | 1024441000 | |
Discovery HS F5 Supelguard Cartridge, 3 μm, L × I.D. 2 cm × 2.1 mm | Merck | 567570-U | HPLC guard column |
Discovery HS F5, 2.1 mm x 100 mm, 3 μm | Merck | 567502-U | HPLC column |
Formic acid | Alchem | 497-94318-50ML | Mobile phase additive |
Glass vials | Perlan Technologies | 5182-0714 | |
HILIC Luna 3 μm, 200A, 100 x 2.0 mm | Shim-Pol | PHX-00D-4449-B0 | HPLC column |
HILIC SecurityGuard Cartridge, 3 μm, 4 x 2.0 mm | Shim-Pol | PHX-AJ0-8328 | HPLC guard column |
Isopropanol | Alchem | 231-AL03262500 | HPLC solvent |
Methanol | Alchem | 696-34966-4X2.5L | HPLC solvent |
Nano-pure water | Merck | 1037281002 | HPLC solvent |
Q Exactive Focus hybrid quadrupole-Orbitrap MS | Thermo Scientific | Q Exactive Focus | Mass Spectrometer |
SeQuant ZIC-cHILIC 3µm,100Å 100 x 2.1 mm | Merck | 1506570001 | HPLC column |
SeQuant ZIC-HILIC Guard Kit 20 x 2.1 mm | Merck | 1504360001 | HPLC guard column |
SPME LC fiber probes, mixed mode | Supelco | prototype fibers | |
UltiMate 3000 HPLC systems | Thermo Scientific | UltiMate 3000 | HPLC system |
Vial inserts (deactivated) | Perlan Technologies | 5181-8872 | |
XSelect CSH C18 3.5μm 2.1x75mm | Waters | 186005644 | HPLC column |
XSelect CSH C18 VanGuard Cartridge 3.5μm, 2.1x5mm | Waters | 186007811 | HPLC guard column |