Chromatography

Liquid chromatography tandem mass spectrometry (LC-MS) was carried out with a Shimadzu Prominence HPLC system and SCIEX 6500 QTRAP mass spectrometer using a Zorbax Extend C18 column (5 μm, 150 × 2.1 mm) at 40°C. Mobile phases were A) 10 mM NH₄OH (pH 10.5) and B) 70% acetonitrile/30% isopropanol/0.1% NH₄OH at a flow rate of 0.5 mL/min. A binary gradient starting at 10% B was linearly increased to 95% B at 4 minutes; then 95% B was held for 0.5 minutes to wash the column. From 4.5 to 4.75 minutes, B was lowered to 10%, and the column was equilibrated in 10% B for 5.25 minutes. The pre-injection and post-injection needle wash was 50% methanol. Column eluent was diverted to waste from 0 to 0.5 minutes and then again from 4.5 to 10 minutes. The total analysis time was 10 min/sample, and injection volumes were 20 μl.

Extraction Protocols:

Samples were precipitated with cold methanol/75 mM NH₄OH/isopropanol (1:1.5:5). The supernatant was dried under Nitrogen gas and reconstituted in 10% methanol to match LC-MS gradient starting conditions.

References

Method derived from the following

[1] Increased flux through the mevalonate pathway mediates fibrotic repair without injury.
Larson-Casey JL, Vaid M, Gu L, He C, Cai GQ, Ding Q, Davis D, Berryhill TF, Wilson LS, Barnes S, Neighbors JD, Hohl RJ, Zimmerman KA, Yoder BK, Longhini ALF, Hanumanthu VS, Surolia R, Antony VB, Carter AB.
J Clin Invest. 2019 Oct 14. pii: 127959. doi: 10.1172/JCI127959. PMID: 31609245

Resources

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