Estimation of Migration-Time and Mobility Distributions in Organelle Capillary Electrophoresis with Statistical-Overlap Theory

Abstract

The separation of organelles by capillary electrophoresis (CE) produces large numbers of narrow peaks, which commonly are assumed to originate from single particles. In this paper, we show this is not always true. Here, we use established methods to partition simulated and real organelle CEs into regions of constant peak density and then use statistical-overlap theory to calculate the number of peaks (single particles) in each region. The only required measurements are the number of observed peaks (maxima) and peak standard deviation in the regions and the durations of the regions. Theory is developed for the precision of the estimated peak number and the threshold saturation above which the calculation is not advisable due to fluctuation of peak numbers. Theory shows that the relative precision is good when the saturation lies between 0.2 and 1.0 and is optimal when the saturation is slightly greater than 0.5. It also shows the threshold saturation depends on the peak standard deviation divided by the region's duration. The accuracy and precision of peak numbers estimated in different regions of organelle CEs are verified by computer simulations having both constant and nonuniform peak densities. The estimates are accurate to 6%. The estimated peak numbers in different regions are used to calculate migration-time and electrophoretic-mobility distributions. These distributions are less biased by peak overlap than ones determined by counting maxima and provide more correct measures of the organelle properties. The procedure is applied to a mitochondrial CE, in which over 20% of peaks are hidden by peak overlap.