Database of two-dimensional polyacrylamide gel electrophoresis of proteins labeled with CyDye DIGE Fluor saturation dye

CyDye DIGE Fluor saturation dye (saturation dye, GE Healthcare Amersham Biosciences) enables highly sensitive 2-D PAGE. As the dye reacts with all reduced cysteine thiols, 2-D PAGE can be performed with a lower amount of protein, compared with CyDye DIGE Fluor minimal dye (GE Healthcare Amersham Biosciences), the sensitivity of which is equivalent to that of silver staining. We constructed a 2-D map of the saturation dye-labeled proteins of a liver cancer cell line (HepG2) and identified by MS 92 proteins corresponding to 123 protein spots. Functional classification revealed that the identified proteins had chaperone, protein binding, nucleotide binding, metal ion binding, isomerase activity, and motor activity. The functional distribution and the cysteine contents of the proteins were similar to those in the most comprehensive 2-D database of hepatoma cells (Seow et al.., Electrophoresis 2000, 21, 1787-1813), where silver staining was used for protein visualization. Hierarchical clustering on the basis of the quantitative expression profiles of the 123 characterized spots labeled with two charge- and mass-matched saturation dyes (Cy3 and Cy5) discriminated between nine hepatocellular carcinoma cell lines and primary cultured hepatocytes from five individuals, suggesting the utility of saturation dye and our database for proteomic studies of liver cancer.     

Proteomic analysis of MCF-7 breast cancer cell line exposed to mitogenic concentration of 17beta-estradiol

Estrogens are powerful mitogens that play a critical role in the onset of breast cancer and its progression. About two-thirds of all breast cancers are estrogen receptor (ER)+ at the time of diagnosis, and the ER expression is the determinant of a tumor phenotype associated with hormone responsiveness. The molecular basis of the relationship between ER expression, (anti)hormonal responsiveness, and breast cancer prognosis is still unknown. To identify the proteins affected by the presence of the hormone we used 2-D-PAGE-based bottom-up proteomics for the study of the proteome of MCF-7 cells of estrogen-responsive breast carcinoma exposed to a mitogenic concentration of 17beta-estradiol (E2) for 12, 18, 24, and 30 h. Differential expression analysis showed significant changes for 12 proteins. These include ezrin-radixin-moesin-binding phosphoprotein of 50 kDa which was previously shown to be directly regulated by E2. Expression profiles of other proteins already implicated in the progression of breast cancer, such as stathmin, calreticulin, heat shock 71 kDa, alpha-enolase are also described. Moreover, it is observed that different unexpected proteins, translation factors, and energetic metabolism enzymes are also influenced by the presence of the hormone.     

Proteome analysis of spermatogonia: identification of a first set of 53 proteins

During spermatogenesis, the various classes of germ cells synthesize proteins necessary for their own functioning and for regulation of the Sertoli cells. However, the nature of these proteins has been little studied, especially in spermatogonia, the germ stem cells. In this study, the electrophoretic patterns of high-resolution, silver-stained, two-dimensional polyacrylamide gels of intracellular spermatogonial protein extracts were studied by computerized gel image analysis. We detected 675 individual spots, some of which we identified by mass spectrometry and database searching. We present here a first set of 53 proteins identified. They include housekeeping proteins never before detected in spermatogonia, ten proteins previously detected in the reproductive tract but not in spermatogonia, including stathmin, a protein previously shown to be involved in cell proliferation and differentiation, and one new testicular protein named translationally controlled tumor protein (TCTP), also known as a growth-related protein. Immunohistochemistry demonstrated that the two latter proteins were indeed highly expressed in spermatogonia in situ, and their possible involvement in spermatogonial division and proliferation is currently under investigation in our laboratory. We conclude that this type of experimental strategy, known as proteomics, is a very powerful way to analyze germ cell proteins comprehensively and should rapidly greatly improve our understanding of spermatogenesis.     

Compatibility of toluidine blue with laser microdissection and saturation labeling DIGE

Tissue fixation and staining protocols for laser microdissection are frequently not fully compatible with subsequent proteomic analysis. We compared the effect of three common histological stains (toluidine blue (TB), hemotoxylin, and hematoxylin and eosin (HE)) on tissue visualization, protein recovery, the saturation labeling reaction, and 2‐D electrophoresis. TB provided the best visualization of colorectal tumor tissue during laser microdissection (LMD) and had a comparable effect on protein recovery and the saturation labeling reaction with hematoxylin, provided a modified 2‐D clean‐up protocol was used. Eosin inhibited both protein recovery and the saturation labeling reaction.