Differential Radioactive Proteomic Analysis of Microdissected Renal Cell Carcinoma Tissue by 54 cm Isoelectric Focusing in Serial Immobilized pH Gradient Gels

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Abstract
We present a proof of principle study, using laser microdissection and pressure catapulting (LMPC) of two clinical tissue samples, each containing approximately 3.8 μg renal cell carcinoma protein and 3.8 μg normal kidney protein respectively from one patient. The study involved separate radio-iodination of each sample with both 125I and 131I, dual inverse replicate sample loading to high resolution 54 cm “daisy chain” serial immobilized pH gradient isoelectric focusing (IPG-IEF) 2D-PAGE gels, co-electrophoretic separation of cross-labeled proteins from different samples, and precision multiplex differential radioactive imaging to obtain signals specific for each sample coelectrophoresed within single gels but labeled with different isotopes of iodine, providing extremely precise intra-gel estimates of the abundance ratio for protein spots from both samples. Twelve multiplexed analytical radioactive SDS-gels from 4 serial IPG-IEF gels provided 24 individual radioactive images for a comprehensive analytical protein multiplex quantification study. A further 12 SDS gels containing 125I-labeled sample were coelectrophoresed with preparative protein amounts obtained from whole tissue sections for the mass spectrometric identification of comigrating proteins. This consumed 125I-labeled sample, and 70 that were >2-fold differentially abundant between the samples and t-test probabilities <0.05. We conclude that this combination of technologies provides excellent quality protein multiplex data for the differential abundance analysis of large numbers of proteins from extremely small samples, and is applicable to a broad range of clinical and related applications. Keywords: immobilized pH gradient • isoelectric focusing • two-dimensional polyacrylamide gel electrophoresis • laser microdissection • cancer