Sources of variability among replicate samples separated by two-dimensional gel electrophoresis

Abstract

Two-dimensional gel electrophoresis (2DE) offers high-resolution separation for intact proteins. However, variability in the appearance of spots can limit the ability to identify true differences between conditions. Variability can occur at a number of levels. Individual samples can differ because of biological variability. Technical variability can occur during protein extraction, processing, or storage. Another potential source of variability occurs during analysis of the gels and is not a result of any of the causes of variability named above. We performed a study designed to focus only on the variability caused by analysis. We separated three aliquots of rat left ventricle and analyzed differences in protein abundance on the replicate 2D gels. As the samples loaded on each gel were identical, differences in protein abundance are caused by variability in separation or interpretation of the gels. Protein spots were compared across gels by quantile values to determine differences. Fourteen percent of spots had a maximum difference in intensity of 0.4 quantile values or more between replicates. We then looked individually at the spots to determine the cause of differences between the measured intensities. Reasons for differences were: failure to identify a spot (59%), differences in spot boundaries (13%), difference in the peak height (6%), and a combination of these factors (21). This study demonstrates that spot identification and characterization make major contributions to variability seen with 2DE. Methods to highlight why measured protein spot abundance is different could reduce these errors.

Keywords: heart; protein; proteomics; reproducibility.

FIGURE 1

Replicates of 2D gels obtained from rat left ventricle. An example of the entire gel is shown in the upper left corner. The box represents the enlarged area of the replicate gels shown in the three remaining images. Numbers and letters designate spots matched across the gels.

FIGURE 2

Example of a failure of spot detection. Spot number 380 was detected in gels 1 and 2 but not in gel 3. The spot does exist in gel 3 but was not detected.

FIGURE 3

Example of a spot abundance difference caused by definition of the spot boundaries. In gel 1 the x dimension is 0.6, but in gels 2 and 3, it is 0.3. In gel number 1, a shoulder of spot number 372 is identified as a part of spot number 373, accounting for the error in intensity between replicates.

FIGURE 4

Example of a spot abundance disparity because of differences in peak height. Spot number 307 has a peak height of 227 in gel 1 and 199 in gel 2 but only 83 in gel 3.