1. HPLC-MS/MS method development for the isolation and identification of inositol
phosphates from C. reinhardtii
Travis Y. Tu, Dr. Bradley Evans, Dr. Mike Naldrett, Dr. Doug Allen, Dr. James
Umen, Dr. Sophie Alvarez
2016 Claremont McKenna College, W.M. Keck Science Department
Introduction Materials and Methods Results
Discussion and Conclusion
Inositol phosphates (IPs) are an extremely important signaling family. Each
of the six hydroxyl groups on an inositol molecule can be phosphorylated to
create 63 different isomers of inositol phosphates, even before considering the
possibility of repeated phosphorylation that would create pyrophosphates(IP7
and IP8)1
. The best studied is Ins(1,4,5)P3 because of its importance as the main
secondary messenger in eukaryotic cells, but many other inositol phosphates
have great biological relevance. IP6 degradation to lower IPs enhance cancerous
activity and IP7 and IP8 have been related plaque formations in Alzheimer’s
patients2,3
. The important thing to realize is that all of these functions are
isomer-specific; simply rearranging the phosphates will cause loss of function.
Yet, it is still extremely difficult to isolate different isomers of IPs from each
other without using expensive chiral columns or time extensive techniques like
iodine peroxidate degradation. There is still no robust, quantitative method to
isolate and identify IP isomers though there have been many attempts ranging
from radioactive labeling to HPLC and mass spectrometry. C reinhardtii has
long been a model organism for studying eukaryotic cells. Recently its potential
as a source of biofuel has also increased interest in studying its lipid
biosynthesis pathways4
. Thus, developing a method to better determine IP
isomer levels in this green algae has far reaching implications.
• WAX column with 250 mM ammonium carbonate and 25% methanol gradient
was the best solvent for eluting higher IPs
• Adding 100 mM ammonium acetate, bicarbonate, or formate SPE elutions or
using IMAC (Fe-NTA columns) removes TCA from C. reinhardtii extractions
• AB Sciex Qtrap 6500+ SelexIonTM
with 2-propanol modifier can separate
different IP isomers standards
• Post-column dilution reduces ion suppression caused by high salt
concentrations required to elute IPs off the WAX column
• Fully assembled method in microHPLC+ post-column dilution+
QTrap6500+ SelexIon allowed for separation and identification of IP
isomers from standards and from C. reinhardtii extractions from IP1
through IP6 based on compensation voltage and detection of IP7 and IP8
• In the future, quantification metrics on microLC+QTrap 6500 with SelexIonTM
will be developed to determine amount of IPs in biological samples. The
method will also be fine-tuned using IP7 and IP8 standards, an increased COV
range, and a more polar modifier such as methanol to achieve further isomer
separation in C. reinhardtii extraction samples.
AcknowledgementsI would like to thank Dr. Bradley Evans, Dr. Mike Naldrett, and Dr. Sophie Alvarez of Donald Danforth Plant Science Center Proteomics
and Mass Spectrometry for advising me this summer. Dr. Mike Naldrett and Dr. Sophie Alvarez for their help in developing the post-column
dilution infusion equipment that allowed me to get past the ion suppression problem . I would also like to extend thanks to my other
advisors, Dr. Doug Allen for helping come up with the ideas to get rid of the TCA in the solid phase extraction and for his weekly assistance
in developing my presentation and to Dr. James Umen for 21GR C. reinhardtii strain, the use of his growth chambers and equipment to
culture our algae and for his input on my presentation as well. I would also like to thank Dr. Inmaculada Couso Lianez for teaching me to
culture and take care of the C. reinhardtii. I would like to thank NSF and the Rose Hills Foundation for Summer Science and Engineering
Research from Claremont McKenna College and the W.M. Keck Science Department for funding. Lastly, I would like to thanks Dr. Larry
Grill and Dr. Nancy Williams for being my readers for my thesis.
References
1. Miranda, S. C., Thomas, M., & Adolfo, S. (2013). Inositol pyrophosphates: between signalling and metabolism. Biochemical
Journal, 452(3), 369-379.
2. McLaurin, J., Franklin, T., Fraser, P. E., & Chakrabartty, A. (1998). Structural transitions associated with the interaction of
Alzheimer β-amyloid peptides with gangliosides. Journal of Biological Chemistry, 273(8), 4506-4515.
3. Shamsuddin, A. M., Vucenik, I., & Cole, K. E. (1997). IP 6: a novel anti-cancer agent. Life sciences, 61(4), 343-354.
4.Hu, Q., Sommerfeld, M., Jarvis, E., Ghirardi, M., Posewitz, M., Seibert, M., & Darzins, A. (2008). Microalgal triacylglycerols as
feedstocks for biofuel production: perspectives and advances. The Plant Journal, 54(4), 621-639.
Clean inositol phosphate extraction1.
Solid Phase Extraction method development
Inositol
Phosphates
Contaminants
TCA
• Wet (MeOH)
• Equilibrate
(MQ H2O) • Load
Sample
• Wash
(25%
MeOH)
• Elute with
100 mM
Ammonium
Acetate,
Bicarbonate
or Formate
Elute with
100mM
Ammonium
Carbonate
Find best solvent and method
Time
(min)
Flow
(microliters/min)
A (%) B (%)
1 0.00 15.00 0.0 100.0
2 4.00 15.00 0.0 100.0
3 10.00 15.00 10.0 90.0
4 24.00 15.00 60.0 40.0
5 30.00 15.00 100.0 0.0
6 34.00 15.00 100.0 0.0
7 40.00 15.00 0.0 100.0
8 70.00 15.00 0.0 100.0
Table 1. Altered gradient for EkspertTM
microLC 200+ QTrap 6500
2.
HPLC method development
After: IP signal is most abundant
Syringe (25%
MeOH)
To Mass
Spec
From
micro-
LC
Post-column dilution apparatus
3. Optimize QTrap 6500 +SelexIonTM
parameters
• Direct infusion of standards
• Test with samples
OH
OH
OH
OHOH
HO
P
P
P
(1, 4, 5)-IP3:
418.9551 Da
1.
2.
3.
(1, 4, 5)-IP3:
418.9551 Da
(1, 4, 5)-IP3:
418.9551 Da
(1, 3, 4)-IP3:
418.9551 Da
(1, 3, 4)-IP3:
418.9551 Da F-IP3: 420.95077 Da
F-IP3: 420.95077 Da