The presence of progesterone receptor (PR) in estrogen receptor (ER)-positive breast cancer is associated with a good prognosis, and indicates that tumors are likely to respond to tamoxifen. However, ER+/PR- tumors respond less well. To reveal the potential molecular mechanism of this phenomenon, we sought to identify differential protein abundances between invasive ductal carcinoma cells from cryopreserved ER+/PR+ and ER+/PR- mammary tumor specimens. Because current proteomics methods are hampered in the examination of most primary human tumor samples by the extreme tissue heterogeneity, we used laser capture microdissection (LCM) to isolate tumor cells and developed a sample pooling strategy to analyze small sample protein lysates. Proteins from LCM-harvested tumors were pooled into four sub-pools from each condition of three tumors/sub-pool, and proteins from respective paired sub-pools were co-electrophoresed by 2-DE using 54-cm IEF over pH 4-9. Abundance ratios were accurately quantified by a differential multiplex radioactive ProteoTope method at low attomole levels ( approximately 3.6 microg protein per labeling reaction, <180 ng per multiplex protein sample per 54-cm gel). Applying this approach, differentially displayed proteins were identified by MS using comigrating non-radioactively labeled tumor proteins. They include decreased cytochrome b5 and transgelin, and more abundant CRABP-II, cyclophilin A, Neudesin, and hemoglobin in ER+/PR+ tumors versus ER+/PR- providing a possible explanation for differential susceptibility against tamoxifen as a result of deregulated cytochrome b5-dependent metabolism. This study demonstrates the potential of ProteoTope and LCM to enable extremely sensitive and precise differential analyses from well-defined primary clinical specimen.

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We present a proof of principle study, using laser microdissection and pressure catapulting (LMPC) of two clinical tissue samples, each containing approximately 3.8 microg renal cell carcinoma protein and 3.8 microg normal kidney protein respectively from one patient. The study involved separate radio-iodination of each sample with both (125)I and (131)I, 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 (125)I-labeled sample were coelectrophoresed with preparative protein amounts obtained from whole tissue sections for the mass spectrometric identification of comigrating proteins. This consumed <40% of the (125)I-labeled sample, and <20% of the (131)I-labeled sample from the respective original 3.8 microg samples. Twenty-nine proteins were identified by mass spectrometry with PMF scores >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.

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