Two time points from each cultivar were analyzed via two-dimensional gel electrophoresis. Two replicates each of Sumner 9/18, Sumner 9/25, Desirable 9/18, and Desirable 9/25 pecan nut extracts were included for comparison. Nut extracts from Desirable 10/2 were also analyzed in response to initial visual comparison between time points via SDS-PAGE. Each biological replicate consisted of protein extracted from three individual nuts. Due to the very low protein content of samples at early time points characterized by the presence of endosperm in the kernel (August 21–September 11) they could not productively be used in the 2D-gel analysis.
Samples were run and computer comparisons were generated by Kendrick Labs, Inc. (Madison, WI, USA), as described previously [30 (link)]. For this study, isoelectric focusing was carried out in a glass tube with an inner diameter of 3.3 mm using 2.0% pH 3–10 isodalt Servalytes (Serva, Heidelberg, Germany), and the gels were silver stained. Spot percentage is equal to a spot integrated density above a background expressed as a percentage of a total density above a background of all spots measured. Protein abundances were compared between developmental time points and between cultivars. Proteins identified as differentially expressed were required to meet the following specific criteria. The spot intensities were required to have a two-fold or greater change of the means with a p-value of less than 0.05 (n = 4). The data from both time points were included in the analysis when comparing the two cultivars and vice versa. For the comparison by developmental time point, a minimum two-fold change was required in both Sumner and Desirable samples individually (n = 2). Conversely, for the comparison by cultivar, a minimum two-fold change was required in both developing and mature time points individually (n = 2). These criteria were also met when using data from the Desirable 10/2 time point in place of the 9/25 time point. The sample in which the spot percentage was deemed the highest must have a spot percentage of at least one-fourth of the mean spot percentage of all the spots on the gel. Spots were visually confirmed for distinguishability and differential expression. Selected spots with significant differences in abundance were cut out and subjected to trypsin digestion to release peptides. Proteins within selected spots were identified by mass spectrometry, as described above.
Samples were run and computer comparisons were generated by Kendrick Labs, Inc. (Madison, WI, USA), as described previously [30 (link)]. For this study, isoelectric focusing was carried out in a glass tube with an inner diameter of 3.3 mm using 2.0% pH 3–10 isodalt Servalytes (Serva, Heidelberg, Germany), and the gels were silver stained. Spot percentage is equal to a spot integrated density above a background expressed as a percentage of a total density above a background of all spots measured. Protein abundances were compared between developmental time points and between cultivars. Proteins identified as differentially expressed were required to meet the following specific criteria. The spot intensities were required to have a two-fold or greater change of the means with a p-value of less than 0.05 (n = 4). The data from both time points were included in the analysis when comparing the two cultivars and vice versa. For the comparison by developmental time point, a minimum two-fold change was required in both Sumner and Desirable samples individually (n = 2). Conversely, for the comparison by cultivar, a minimum two-fold change was required in both developing and mature time points individually (n = 2). These criteria were also met when using data from the Desirable 10/2 time point in place of the 9/25 time point. The sample in which the spot percentage was deemed the highest must have a spot percentage of at least one-fourth of the mean spot percentage of all the spots on the gel. Spots were visually confirmed for distinguishability and differential expression. Selected spots with significant differences in abundance were cut out and subjected to trypsin digestion to release peptides. Proteins within selected spots were identified by mass spectrometry, as described above.
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