BAF_Protocol_015_ 3-NPH derivatization of short chain fatty acid
Nicholas Sherman
Abstract
Short Chain Fatty Acid derivitization before MS analysis.
Steps
Samples
Refer to BAF_Protocol_008 Metabolomics: Soluble Metabolite Extraction for soluble metabolite extraction of a sample of interest and dry the extracted metabolites.
Derivatization reagents: prepare all fresh
Reagent powder storage: EDC is stored at -20C, all others are at room temperature.
Prepare working solutions on small glass botles.
250 nM 3-NPH solution as derivatization agent in 50% methanol:
--> dissolve 47.4 mg of 3-NPH in 1 mL of 50% methanol
2 mg/mL BHT in 100% methanol:
--> dissolve 2 mg in 1 mL methanol.
150 mM EDC (stored at -20C) in 100% methanol:
--> dissolve 28.75 mg in 1 mL methanol.
7,5% pyridine in 75% methanol.
--> add 750 uL in 9.25 mL of 75% methanol.
Standards and standard curve
Prepare a stock solution of each standard at 10 mg/mL in 75% MeOH. Use the stock solution to generate a 1000 ug/mL
Prepare standard curve:
| A | B | C | D | E | F |
|---|---|---|---|---|---|
| Workingsolution (ug/mL) | Workingsolution vol (uL) | Diluent vol (uL) | Final Conc. (ug/mL) | ng/mL | Dilution level |
| 1000 | 1000 | 0 | 1000 | 1E6 | |
| 100 | 100 | 900 | 100 | 100,000 | L1 |
| 10 | 100 | 900 | 10 | 10,000 | L2 |
| 1 | 100 | 900 | 1 | 1000 | L3 |
| 0.1 | 100 | 900 | 0.1 | 100 | L4 |
| 0.01 | 100 | 900 | 0.01 | 10 | L5 |
| 0.001 | 100 | 900 | 0.001 | 1 | L6 |
Derivatization reaction
Solubilize metabolite extracts in 100µL of 75% methanol
Add 50µL of each into a clean new Eppendorf tube
Add 50µL of each standard curve level into a clean new Eppendorf tube
To each sample or standard add:
50µL of 150 mM EDC,
50µL of 7,5% pyridine,
Incubate in thermomixer at 30 ºC
To quench the reaction, add 50µL of BHT
Dilute with 250µL of water making a total of volume of 500µL and to achieve 40% methanol at final sample.
Samples are ready to be analyzed by LC-MS/MS. Use 40% methanol as blank runs.
LC-MS/MS parameters - Vanquish UPLC
Column: Acquity UPLC BEH C-18 1.7 um 2.1 x 150 mm (Waters)Solvent A: 0.1% FA in water
Solvent B: 0.1% FA in 90% methanol
Flow = 0.250 ml/min.
Gradient:
0-1 min 50%B; 1-5 min 70% B; 5-8 min 98% B; 8-13 98% B; 13-13.1 min 15%B.
Mass spectrometer parameters - Orbitrap ID-X
Full scan parameter MS1 Res = 120,000Range = 67-1000RF Lens = 60AGC = 25%Max IT (ms)= 50Polarity = Negative PRM parameters: CE=30Res: 50,000Isolation window: 1.5AGC: 50MaxIT (ms): 86Polarity = NegativeTargeted masses:
Analyze data using Skyline.
