Yongchao Li (Presenter)
University of Illinois at Chicago
Bio: I am Yongchao Li and got my M.D. from China at 2004. After my medical school, I am very interesting in translational research so that I came to US to pursue Ph.D degree at U of Illinois at Chicago College of pharmacy major in Analytical chemistry. During the Ph.D training, I got lots of chance to do some translational and clinical research projects at University and industry. I have the experience in translational and clinical mass spec lab under the requirement of CLIA and CAP and capability of training the staff about the LC-MS method development and validation.
Authorship: Yongchao Li1, Richard B. van Breemen1
1. University of Illinois College of Pharmacy, 833 S Wood St, Chicago, IL, 60612
| Short Abstract After identification of the biomarker and treatment target of asthma by LC-MS based lipidomics and we develop and validate LC-MS based Autotaxin functional assay and screen the inhibitors from mixture (botanical extracts) by PUF-LCMS. |
Long Abstract
Objective: After identification of the biomarker and treatment target of asthma by LC-MS based lipidomics and we develop and validate LC-MS based Autotaxin functional assay and screen the inhibitors from mixture (botanical extracts) by PUF-LCMS.
Methods: LPA levels were determined using liquid chromatography (LC) and tandem mass spectrometry with an ABI-5500 Q-TRAP hybrid triple quadrupole/ion trap mass spectrometer (MS) coupled with an Agilent 1100 liquid chromatography system. Lipids were separated using methanol-water-HCOOH, 79:20:0.5, vol/vol/vol with 5 mM NH4COOH as solvent A and methanol-acetonitrile-HCOOH, 59:40:0.5, vol/vol/vol with 5 mM NH4COOH as solvent B. LPA molecular species were analyzed in negative ionization mode with declustering potential and collision energy optimized for LPA.
To verify that this new PUF-LCMS assay could detect ATX ligands, the known ligands PF-8380, S32826, GWJ-23, and BrP-LPA were screened. Using denatured ATX as a negative control, LC-MS analysis of the ultrafiltrates after incubation, washing, and release of the same known ATX ligands indicated that these compounds exhibited little or no non-specific binding to denatured ATX or the PUF apparatus. Based on the LC-MS peak enhancement for the known ligands of ATX relative to the corresponding peaks in the negative controls, all of the ligands produced signals indicative of positive hits in the PUF-LCMS assay.
A functional ATX enzyme activity assay was developed and validated based on LC-MS/MS quantification of the reaction product, C16:0 LPA formed from the enzymatic release of choline from C16:0 LPC. The unnatural phospholipid, C17:0 LPA, was used an internal standard.
Results and Conclusion: The PUF-LCMS assay and functional ATX enzyme activity assay were fully developed and validated. Total LPA levels in BAL fluid were increased by allergen challenge in mild asthmatics, mainly due to the elevation of LPA 22:5 and 22:6 ranging from 2- to 100-fold increases. The assay was successfully applied to the screening of botanical extracts, and the natural product glabridin, from G. glabra, was identified through dereplication.
References & Acknowledgements:
References:
1. van Breemen, R.B., et al., Pulsed ultrafiltration mass spectrometry: a new method for screening\ combinatorial libraries. Anal Chem\, 1997. 69\(11\): p. 2159-64\.
2. Choi, Y., et al., Screening natural products for inhibitors of quinone reductase-2 using\ ultrafiltration LC-MS. Anal Chem\, 2011. 83\(3\): p. 1048-52\.
Acknowledgments:
We thank all labmates from Dr. John W Christman's lab.
The work was supported by the National Cancer Institute and from grant R01AT007659 (RBvB) from the Office of the Director and the National Center for Complementary and Integrative Health of the National Institutes of Health.
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