HPLC and MS/MS Method for the Separation and Identification of Inositol Phosphates in C. Reinhardtii
Graduation Year
2016
Date of Submission
12-2015
Document Type
Campus Only Senior Thesis
Degree Name
Bachelor of Arts
Department
W.M. Keck Science Department
Second Department
Biochemistry
Reader 1
Nancy Williams
Reader 2
Bradley Evans
Reader 3
Larry Grill
Terms of Use & License Information
Rights Information
© 2015 Travis Y. Tu
Abstract
Inositol phosphates (IPs) have important cellular functions from teleomere maintenance (IP7 and IP8) to Ca2+ signaling pathways (1, 4, 5-IP3). Yet there is no robust, quantitative method to separate all the inositol phosphate isomers from IP1 to IP8. Four findings contributed heavily towards the development of a robust, quantitative IP isomer separation and identification method on the EskpertTM MicroLC 200+ QTrap 6500 system with a SelexIonTM DMS attachment. 1) TCA from inositol phosphate algal extractions was removed by elution with 100 mM ammonium carbonate, ammonium formate, or ammonium bicarbonate or by immobilized metal affinity chromatography (Fe-NTA columns). 2) A 250 mM ammonium carbonate and 25% methanol gradient was run on a weak anion exchange column to separate all the inositol phosphates from each other (IP1 through IP8. 3) Using ten different inositol phosphate isomer standards and fluoro- IP3 as an internal standard for future quantitation and recovery studies, isomer separation was obtained using SelexIonTM DMS with a 2- propanol modifier. 4) Ion suppression of inositol phosphate signals caused by 250 mM ammonium carbonate can be alleviated with a post-column dilution. The final assembled EskpertTM MicroLC 200+ QTrap 6500 system with a SelexIonTM DMS attachment and post-column dilution method was able to separate out IP isomers from IP1 to IP6 and detect IP7 and IP8. Once further optimized using the full compensation voltage range and a more polar modifier such as methanol, this method will allow the lipid biosynthesis pathways of C. reinhardtii, a promising candidate for algal biofuels, to be better studied.
Recommended Citation
Tu, Travis Y., "HPLC and MS/MS Method for the Separation and Identification of Inositol Phosphates in C. Reinhardtii" (2016). CMC Senior Theses. 1306.
https://scholarship.claremont.edu/cmc_theses/1306
This thesis is restricted to the Claremont Colleges current faculty, students, and staff.
