Immobilized pH Gradient Two-dimensional Gel Research Articles

The exoproteomes of clonally related Staphylococcus aureus strains are diverse

Several studies have shown that protein expression patterns vary in unrelated bacterial strains due to genomic plasticity and gene regulation, resulting in enhanced heterogeneity in the infection potential. However, exoprotein expression patterns of closely related clonal strains have not been well characterized. Here, we used medium-range (pH 4–7) immobilized pH gradient–two-dimensional gel electrophoresis to investigate the exoproteome from closely related Staphylococcus aureus clonal isolates. Interestingly, we found that, under identical in vitro experimental conditions, a number of protein spots were uniquely present in samples from each clonal isolate irregardless of the similarity of the genotype and the same virulence gene profile. Only a few abundant invariant proteins were found among identical genotypic isolates. Our results clearly shown that heterogeneity in the exoproteome was present even among clonally related strains. We suggest that this heterogeneity may contribute to the degree of virulence even within one clonal genotype. The heterogeneity in the exoproteome of closely related S. aureus strains observed in the current study postulates that pre-existing antibodies are not very protective during recurrent infection with the same strain. Therefore, our findings underscore the importance of taking all clonally related strains into account during proteome analyses.

Differential expression of secretagogin and glucose-related protein 78 in colorectal carcinoma: a proteome study

To identify the differentially expressed proteins or peptides and potential biomarkers of tumorigenesis for colorectal cancers. Immobilized pH gradient two-dimensional gel electrophoresis (2-DE) was used to separate and obtain the differentially expressed protein spots between colorectal cancers and matched normal mucosa. Liquid chromatography/mass spectrometry (LC-MS/MS) was used to characterize these proteins. Selected candidate proteins were further studied by Western blot, semi-quantitative RT-PCR and immunohistochemical staining. Thirty-five protein spots showed marked expression changes (more than 5-fold) in colorectal carcinoma compared to normal mucosa. Fifteen proteins were up regulated and 20 were down regulated. Fourteen of these proteins were identified by tandem mass spectrometry, among which secretagogin (SCGN) was down-regulated and glucose-related protein (GRP) 78 was up-regulated in the tumors. The SCGN down-regulation was further supported by Western blot and RT-PCR analyses. Immunohistochemistry revealed that SCGN was strongly expressed in neuroendocrine cells of the colonic crypts and 53 of 54 (98%) neuroendocrine tumors. At protein level, although GRP78 was up regulated in colorectal carcinoma, there was no difference in the mRNA expression level between the tumor and paired normal mucosa. The 2-DE combined with MS is a powerful tool for screening potential tumor biomarkers. The differentially expressed candidate proteins identified by 2-DE may be of significance in understanding the tumorigenesis of the colon cancer. SCGN is a potential biomarker for neuroendocrinal differentiation. GRP78 up-regulation in colorectal carcinomas may be related to its post-translational modification.

Proteome Changes in Ovarian Epithelial Cells Derived from Women with BRCA1 Mutations and Family Histories of Cancer

Malignant transformation of the ovarian surface epithelium (OSE) accounts for most ovarian carcinoma. Detection of preneoplastic changes in the OSE leading to overt malignancy is important in prevention and management of ovarian cancer. We identified OSE proteins with altered expression derived from women with a family history (FH) of ovarian and/or breast cancer and mutations in the BRCA1 tumor suppressor gene. Proteins from SV-40-transformed FH-OSE cell lines and control OSE lines derived from women without such histories (non-family history) were separated by two-dimensional PAGE. Gels were analyzed, a protein data base was created, and proteins were characterized according to their molecular weight, isoelectric point, and relative abundance. Mass spectrometry was performed on tryptic protein digests, and data bases were searched for known proteins with the same theoretical tryptic peptide masses. Several proteins showed altered expression in the FH-OSE cells. Beta-tubulin and to a lesser extent ubiquitin carboxyl-terminal hydrolase and glyoxalase 1 appeared to be up-regulated. In contrast, proteins suppressed in FH lines include the 27-kDa heat shock protein, translationally controlled tumor protein, and several proteins associated with actin modification such as actin prepeptide, F-actin capping protein alpha subunit, and cofilin. Sequencing of several cofilin gel spots revealed phosphorylation of serine 3, a post-translational modification associated with decreased actin binding and cytoskeletal reorganization. Two-dimensional Western blots probed with cofilin antibody showed multiple protein spots with isoelectric points of 6-9 pH units. Blots of one-dimensional gels showed a significant reduction in cofilin expression in three FH lines when compared with three non-family history lines (p < or = 0.05). Identification of these and other OSE proteins may be useful in detecting changes suggestive of increased risk of developing preneoplastic disease and defining the possible role(s) of the BRCA1 gene in regulation of OSE cell function.

Characterization of Gel-separated Glycoproteins Using Two-step Proteolytic Digestion Combined with Sequential Microcolumns and Mass Spectrometry

Protein glycosylation can be vital for changing the function or physiochemical properties of a protein. Abnormal glycosylation can lead to protein malfunction, resulting in severe diseases. Therefore, it is important to develop techniques for characterization of such modifications in proteins at a sensitivity level comparable with state-of-the-art proteomics. Whereas techniques exist for characterization of high abundance glycoproteins, no single method is presently capable of providing information on both site occupancy and glycan structure on a single band excised from an electrophoretic gel. We present a new technique that allows characterization of low amounts of glycoproteins separated by gel electrophoresis. The method takes advantage of sequential specific and nonspecific enzymatic treatment followed by selective purification and characterization of the glycopeptides using graphite powder microcolumns in combination with mass spectrometry. The method is faster and more sensitive than previous approaches and is compatible with proteomic studies.

Mapping and identification of interferon gamma-regulated HeLa cell proteins separated by immobilized pH gradient two-dimensional gel electrophoresis.

Interferon gamma (IFN-gamma) is a potent immunomodulatory lymphokine, secreted by activated T-lymphocytes and NK-cells during the cellular immune response. Actions of IFN-gamma are mediated through binding to the IFN-gamma-receptor, present on most cells, and the subsequent activation of a great magnitude of IFN-gamma responsive genes has been reported previously. Our goal is to identify and map IFN-gamma-regulated HeLa cell proteins to the two-dimensional polyacrylamide gel electrophoresis with the immobilized pH gradient (IPG) two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) system. A semiconfluent layer of HeLa cells was grown on tissue culture plates, and changes in protein expression due to 100 U/mL IFN-gamma were investigated at different periods after treatment, using pulse labeling with [35S]methionine/cysteine in combination with 2-D PAGE (IPG). The identity of eight protein spots was elucidated by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), and several variants of the IFN-gamma-inducible tryptophanyl-tRNA synthetase (hWRS) were detected by immunoblotting.

Immobilized pH gradient two-dimensional gel electrophoresis and mass spectrometric identification of cytokine-regulated proteins in ME-180 cervical carcinoma cells.

Two-dimensional (2-D) polyacrylamide gel electrophoresis combined with mass spectrometry is a powerful combination of technologies that allows high resolution separation of proteins and their rapid identification. Immobilized pH gradient (IPG) first-dimensional gels have several advantages over carrier ampholyte isoelectric focusing, including a high degree of reproducibility, good protein spot resolution, and a selection of pH range. Here we demonstrate the utility and efficacy of combining IPG 2-D gel electrophoresis with mass spectrometry to identify interferon-gamma- (IFN) and tumor necrosis factor (TNF)-regulated proteins in ME-180 cervical carcinoma cells. Three cytokine-regulated proteins have been identified, using imidazole-zinc-stained preparative IPG 2-D gels and in-gel tryptic digestion followed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry for determination of peptide masses and sequences: 1) triosephosphate isomerase, a glycolytic pathway enzyme, 2) proteasome subunit C3, which is important in protein degradation, and 3) Ran, a GTP-binding protein important in cell cycle regulation, protein import into the nucleus, and RNA export from the nucleus.