Evaluation of individual particle capillary electrophoresis experiments via quantile analysis

Abstract

The number of particles in a sample heavily influences the shape of the distribution describing the corresponding individual particle measurements. Selecting an adequate number of particles that prevents biases due to sample size is particularly difficult for complex biological systems in which statistical distributions are not normal. Quantile analysis is a powerful statistical technique that can rapidly compare differences between multiple distributions of individual particles. This report utilizes quantile analysis to show that the number of events detected affects the mobility distributions for rat liver and mouse liver mitochondria, sample individual particles, when analyzed via capillary electrophoresis with laser-induced fluorescence. When the mitochondrial sample is small (e.g. <78), there are not enough events to obtain statistically relevant mobility data. Adsorption to the capillary surface also significantly affects the mobility distribution at a small number of events in uncoated and dynamically coated capillaries. These adsorption effects can be overcome when the mitochondrial load on the capillary is sufficiently large (i.e. >609 and >1426 events for mouse liver on uncoated capillaries and rat liver on dynamically coated capillaries, respectively). It is anticipated that quantile analysis can be used to study other distributions of individual particles, such as nanoparticles, organelles, and biomolecules, and that distributions of these particles will also be dependant on sample size.

Determining under‐ and oversampling of individual particle distributions in microfluidic electrophoresis with orthogonal laser‐induced fluorescence detection

“…While the electrophoretic mobility histograms also appear similar to the previous CE reports [17,28], it can be difficult to make a detailed comparison of a large number of distributions using histograms, particularly when these distributions cannot be easily mathematically described. Thus, a better method for comparing mobility distributions was developed for this purpose.…”

“…2, which resembles the electropherograms of individual mitochondrial previously obtained on CE [17,28] and microfluidic devices [15]. In order to prevent unwanted adsorption to the glass microfluidic device, a buffer containing 0.2% w/w PVA was used.…”

“…Previous reports by this laboratory have suggested that the number of detected particles and interactions with the fused silica surface play a critical role in the electro-phoretic mobility distributions of mitochondria in CE [17]. It is reasonable to assume that separations of mitochondria on glass microfluidic devices will behave in a similar fashion.…”

“…In order to prevent unwanted adsorption to the glass microfluidic device, a buffer containing 0.2% w/w PVA was used. This PVA dynamic coating has allowed for separation of mitochondria on both capillaries [17] and glass microchips [15]. In addition, this coating reduces strong adsorption of mitochondria, thereby reducing the number of events that were the result of carryover from a previous sample.…”

“…Thus, a better method for comparing mobility distributions was developed for this purpose. q-q plots are a very effective means of comparing the differences in location, shape, and skew of a large number of electrophoretic mobility distributions [17]. A quantile is defined as the value at a given fraction of a distribution (i.e., the 0.5 quantile is the median).…”