TITLE: F5 Targeted profiling method for metabolomics
AUTHORS: Megan Danielewicz

[DESCRIPTION]
This protocol details the preparation of reagents and samples and extraction of plasma.

[STEPS]
SECTION: Extraction protocol
3. Add sample into 1.5 mL eppendorf tube:
SECTION: Extraction protocol
4. Add 1000 µL of methanol (preferably ice cold)
SECTION: Extraction protocol
6. Vortex the sample/ solvent mixture for 10 s.
SECTION: Extraction protocol
8. Centrifuge the sample/ solvent mixture at 10000 rcf, 10 min, 8 °C.
SECTION: Extraction protocol
10. Optional step: Chill  4 °C for 120 min for any further precipitation of proteins.


SECTION: Extraction protocol
13. Centrifuge the sample/solvent mixture again at 15000 rcf, 10 min (can use a centrifuge filter). 
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15. Inject 5 µL of the supernatant for LC-MS analysis.



SECTION: Preparation of reagents and solutions
1. Mobile Phase and Sample prep

The instructions for preparing each reagent/solution are provided below:

SECTION: Preparation of reagents and solutions
1.1. Mobile phase A (0.1% formic acid in water):

Mix 1 mL of formic acid with 999 mL of water in a 1 L bottle.

SECTION: Preparation of reagents and solutions
1.2. Mobile phase B (0.1% formic acid in acetonitrile):

Mix 1 mL of formic acid with 999 mL of methanol in a 1 L bottle.

SECTION: Preparation of reagents and solutions
1.3. Needle rinse (1:1:1:1 water/methanol/iso-propanol/acetonitrile):

Mix 250 mL of water, 250 mL of methanol, 250 mL of isopropanol and 250 mL of acetonitrile in a 1L bottle.



SECTION: Sample and Control Preparation
2. The instructions for preparing the double blank, blank and QC samples are listed below:
SECTION: Sample and Control Preparation
2.1. CIL QRESS Internal standard stock solution prep

QReSS vial 1 Stock: Add 1 mL of 50% methanol via a needle to the stoppered vial to reconstitute (do not remove stopper prior to reconstitute).
QReSS vial 2 Stock: Add 1 mL of 50% methanol via a needle to the stoppered vial to reconstitute (do not remove stopper prior to reconstitute).


Take 10.25 µL of each QReSS vial 1 and vial 2 and add 979.5 µL of methanol.
SECTION: Sample and Control Preparation
2.2. Double blank sample:

Pipet 1 mL 1:1 water/methanol into an autosampler vial.
SECTION: Sample and Control Preparation
2.4. Pooled sample for method development:

Sample extracts containing internal standards can be used.
SECTION: Sample and Control Preparation
2.3. Blank sample:

Add 50 µL water to 390 µLmethanol and 10 µL of internal standard stock solution.
SECTION: Extraction protocol
11. Dry down supernatant via speedvac or nitrogen evaporation (preferred)
SECTION: Extraction protocol
12. Reconstitute in 100 µL .

SECTION: Gradient conditions
16. 
 

 

 

SECTION: Source Conditions
17. 
 

 
MRM list can be provided upon request.
QRESS MRM and RT can be provided upon request.
SECTION: Extraction protocol
3.1.  Mouse organ - 100 mg minimum (or closest you can come to that)
SECTION: Extraction protocol
3.2. Plasma – 100 uL minimum - Make sure the plasma is settled in the bottom of the tube but not on the walls of the tube.
SECTION: Extraction protocol
3.3.  Bacterial supernatant/spent/fresh media – 100 uL -- may need to dilute later
SECTION: Extraction protocol
3.4. Brine or Food - 100 uL or 100 mg
SECTION: Extraction protocol
3.5. ALWAYS MAKE – A Double blank – An HPLC vial with 1 mL Mobile phase A (no sample processing)
SECTION: Extraction protocol
3.6. ALWAYS MAKE – An extraction blank -- process an empty tube alongside your samples
SECTION: Extraction protocol
3.7. ALWAYS MAKE – An ISTD blank – a blank with only an ISTD spike processed alongside your samples  Add 20 uL of “QRESS ISTD spiking stock”. Always want to add it before extraction to normalize for what happens to the sample during extraction
SECTION: Extraction protocol
4.1. Add  + 20 µL of QReSS IS spiking stock solution
SECTION: Extraction protocol
7. Chill for 10+ min in freezer. Can chill overnight.
SECTION: Extraction protocol
9. Transfer supernatant to fresh labeled eppendorf.
SECTION: Extraction protocol
14. Transfer 50 uL of solution to fresh labeled HPLC vial with insert.

SECTION: Extraction protocol
5. If your sample is a solid, perform beat beating for 1 min, chilling every 30 seconds.  After bead beating chill for 30 min.
SECTION: Extraction protocol
9.1. If you are using an aliquot of this for the NPH derivatization, take it now!!! 
SECTION: Extraction protocol
9.2. NPH aliquot:  Add 50 uL to a second labeled Eppendorf.