| Popis: |
New pipelines are required to automate the quantitation of emerging high-throughput electrophoretic (EP) assessment of DNA damage, or proteoform expression in single cells. EP cytometry consists of thousands of Western blots performed on a microscope slide-sized gel microwell array for single cells. Thus, EP cytometry images pose an analysis challenge that blends requirements for accurate and reproducible analysis encountered for both standard Western blots and protein microarrays. Here, we introduce the Summit algorithm to automate array segmentation, peak background subtraction, and Gaussian fitting for EP cytometry. The data structure storage of parameters allows users to perform quality control on identically processed data, yielding a ~6.5% difference in coefficient of quartile variation (CQV) of protein peak area under the curve (AUC) distributions measured by four users. Further, inspired by investigations of background subtraction methods to reduce technical variation in protein microarray measurements, we aimed to understand the trade-offs between EP cytometry analysis throughput and variation. We found an 11%-50% increase in protein peaks that passed quality control with a subtraction method similar to microarray "average on-boundary" versus an axial subtraction method. The background subtraction method only mildly influences AUC CQV, which varies between 1% and 4.5%. Finally, we determined that the narrow confidence interval for peak location and peak width parameters from Gaussian fitting yield minimal uncertainty in protein sizing. The AUC CQV differed by only ~1%-2% when summed over the peak width bounds versus the 95% peak width confidence interval. We expect Summit to be broadly applicable to other arrayed EP separations, or traditional Western blot analysis. |
