A New, Rapid Method for the Quantification of Dolichyl Phosphates in Cell Cultures Using TMSD Methylation Combined with LC–MS Analysis

Materials and reagents

1. Pipette tips, 50–1,000 μL (Eppendorf, catalog number: 613-3505)

2. Gastight syringe, 5 mL (Hamilton, catalog number: 81517)

3. Pipette tips Combitips advanced 25 mL (Eppendorf, catalog number: EP 0030089472)

4. Round bottom threaded glass tubes (PYREX, catalog number: 9447161)

5. Pasteur pipettes, soda-lime glass (Brand, Wertheim, Germany, catalog number: 747715)

6. Micro inserts, Neochrom (Neolab, catalog number: 7-0635)

7. LC vials, Neochrom (Neolab, catalog number: EC-1184)

8. Threaded screw caps with Teflon Liner (Corning, catalog number: 9998-15)

9. Green nitrile gloves (TouchNtuff, catalog number: 92600)

10. Ammonium acetate (AmAc), eluent additive for LC-MS, LiChropur (Sigma-Aldrich, Merck KGaA, catalog number: 73594)

11. Formic acid (FA), eluent additive for LC-MS (Honeywell, catalog number: 56302)

12. Ammonium bicarbonate (Sigma-Aldrich, Merck KGaA, catalog number: 09830)

13. Acetonitrile, LC-MS grade (Fisher Chemical, catalog number: 10616653)

14. Methanol, LC-MS grade (Fisher Chemical, catalog number: 10532213)

15. Isopropanol, LC-MS grade, Optima (Fisher Chemical, catalog number: A461212)

16. Water, LC-MS grade, Optima (Fisher Chemical, catalog number: 10505904)

17. Dichloromethane (Sigma-Aldrich, Merck KGaA, catalog number: 270997)

18. Dolichol 13–21 phosphate mixture (DolP standard hereafter) (Sigma-Aldrich, Merck KGaA, catalog number: 900201X)

19. Dodecaprenyl phosphate (PolP C60) (CymitQuimica, catalog number: 48-62-1060)

20. Mini-BeadBeater q mill beads (0.5 mm) (BioSpec Products, catalog number: 11079105)

21. Potassium hydroxide pellets (KOH) (Sigma-Aldrich, Merck KGaA, catalog number: 21473)

22. Trimethylsilyldiazomethane (TMSD) (Sigma-Aldrich, Merck KGaA, catalog number: 362832)

23. Acetic acid ACS grade (Sigma-Aldrich, Merck KGaA, catalog number: 33209)

24. HeLa/Fibroblast cells (~1 M cells)

25. S. cerevisiae cells (~0.8 OD)

26. Acquity UPLC CSH C18 Column, 1.7 μm, 1 mm × 150 mm (Waters, catalog number: 186005294)

27. Acquity UPLC CSH C18 VanGuard Pre-column, 1.7 μm, 2.1 mm × 5 mm (Waters, catalog number: 186005303)

28. For the preparation of liquid chromatography, mobile phase A (Solvent A) and mobile phase B (Solvent B), and wash solvents, please see Recipes.

Solutions

1. 15 M KOH (see Recipes)

2. 10 M AmAc (see Recipes)

3. Solvent A: acetonitrile/water 60:40 (v/v) with 0.1 % FA and 10 mM AmAc (see Recipes)

4. Solvent B: isopropanol/water 90:10 (v/v) with 0.1 % FA and 10 mM AmAc (see Recipes)

5. Wash solvent: dichloromethane:methanol:water (3:48:47, v/v/v) (see Recipes)

6. Dichloromethane:methanol mixture (6.5:5.2, v:v) (see Recipes)

7. 155 mM ammonium bicarbonate buffer (see Recipes)

Recipes

1. 15 M KOH

   Accurately weigh 8.4 g of KOH pellets into a 100 mL volumetric flask. Add LC-grade water dropwise with intermittent slow vortex to dissolve KOH. Be careful, as an exothermic reaction will occur, and the reaction flask will be hot. After complete dissolution, dilute the solution to the mark. Use a glass bottle with a PTFE lid for storage of the KOH solution.




2. 10 M AmAc

   Take the 10 mL volumetric flask along the glass stopper and rinse both twice with MS-grade methanol. Dry them carefully under Argon. Transfer accurately weighed 7.708 g of LC-MS grade AmAc to the volumetric flask. To dissolve the solid, add distilled water in steps of approximately 100 µL, followed by sonication. Once all solids are dissolved, fill the volumetric flask to the etched line. This stock can be stored at 4 °C for up to six months.




3. Solvent A: acetonitrile/water 60:40 (v/v) with 0.1% FA and 10 mM AmAc

   Prepare 1,000 mL of solvent mixture of acetonitrile/water 60:40 (v/v) and add 1 mL of formic acid and 1 mL of 10 mM AmAc stock. Mix solvent mixture thoroughly and degas for 5 min in an ultrasonic water bath sonicator.




4. Solvent B: isopropanol/water 90:10 (v/v) with 0.1% FA and 10 mM AmAc

   Prepare 1,000 mL of solvent mixture of isopropanol/water 90:10 (v/v) and add 1 mL of FA and 1 mL of 10 mM AmAc stock. Mix solvent mixture thoroughly and degas for 5 min in an ultrasonic water bath sonicator.




5. Wash solvent: dichloromethane:methanol:water (3:48:47, v/v/v)




6. Dichloromethane:methanol mixture (6.5:5.2, v:v)




7. 155 mM ammonium biocarbonate buffer

Transfer accurately weighed 1.2 g of ammonium bicarbonate into a 100 mL volumetric flask. Dissolve in distilled water and make up to the mark with distilled water. This stock can be stored at 4 °C for up to six months.

Equipment

1. Eppendorf Multipette E3 (Eppendorf, catalog number: VB-1748)

2. Eppendorf Research plus pipette 100–1,000 μL (Eppendorf, catalog number: 3123000063)

3. Ultimate 3000 LC system (Dionex, Thermo Fisher Scientific) system coupled to a Q-Exactive HRMS (Thermo Scientific)

4. Vortexer, SI Vortex-Genie 2 (Scientific Industries, catalog number: 15547335)

5. Nitrogen evaporator (TurboVap, catalog number: 416200)

6. Syringe, gastight HamiltonTM1700 Series 100 μL (Sigma-Aldrich, MerckKGaA, catalog number: 111HAM201050)

7. Refrigerated centrifuge, Heraeus Megafuge 16R (Thermo Scientific, catalog number: 75004271)

8. Ultrasonic water bath sonicator (Bandelin sonorex)

9. Water bath, LSB Aqua Pro (Grant Instruments, catalog number: LSB12)

Software

1. XCalibur software 3.2 (Thermo Fisher Scientific)

2. Online EnviPat R package Tool (available at https://www.envipat.eawag.ch/, Accessed: 21 August 2019)

Procedure