PT  - JOURNAL ARTICLE
AU  - Shefali Lathwal
AU  - Raaisa Raaisa
AU  - Tiago C. Alves
AU  - Richard G. Kibbey
AU  - Abhishek K. Jha
AU  - Graeme F. Mason
TI  - Optimization of Experiment Design for Mass Spectrometric Isotopic Labeling Kinetics
AID  - 10.1101/331520
DP  - 2018 Jan 01
TA  - bioRxiv
PG  - 331520
4099  - http://biorxiv.org/content/early/2018/05/27/331520.short
4100  - http://biorxiv.org/content/early/2018/05/27/331520.full
AB  - Determination of metabolic fluxes by measurement of time-dependent sampling of isotopic enrichments during the administration of labeled substrates provides rich information. Because such experiments are resource-intensive and frequently push the limits of sensitivity of the measurement techniques, optimization of experiment design can improve feasibility with respect to financial and labor costs, time to completion, and increase precision and accuracy of the results. Here we used a previously published set of data acquired in cultured insulinoma cells to evaluate contributions to the sensitivity and variability of the rate of citrate synthase (CS). Specifically, we calculated changes in uncertainty in CS if sample times were dropped or new ones were added, and we observed that some sampling times can be dropped with little effect, while improvements can be made with a strategic choice of when to add samples. We measured the contributions of data sampled at different times on the sensitivity of CS, finding that CS had greater sensitivity at early time points. We tested the concept that if two estimated parameters are correlated significantly, then refining one might constrain the other. In this case, the rate of Beta-oxs was found to be correlated with CS, and narrower variability in Beta-ox did indeed improve the sensitivity of CS. The tests described here might be applied at the initial design stage and then after a pilot phase to improve sensitivities of targeted fluxes and the reduction of materials, time, labor, and other experimental resources. The correlation analyses can be used to consider what orthogonal measurements might be beneficial for further improvement of measurements. While this study used a specific example of a set of time-dependent kinetic isotopic measurements, the results illustrate some generalizable behaviors that can be tested in other experimental systems.Author ContributionsShefali Lathwal designed PollyPhi Absolute(R), ran calculations, wrote manuscript. Raaisa designed PollyPhi Absolute(R), ran calculations, wrote manuscript. Tiago C. Alves acquired mass spectrometric data, contributed to manuscript. Richard Kibbey oversaw acquisition of mass spectrometric data, contributed to manuscript. Abhishek K. Jha oversaw development of PollyPhi Absolute, contributed to manuscript. Graeme F. Mason advised on design of PollyPhi Absolute, planned experiment design tests, wrote manuscript.