Proteome differences between male and female fetal cells in amniotic fluid.

In mammals, sex development is genetically and hormonally regulated. The process starts with the establishment of chromosomal structures (XY or XX), followed by the expression of sex-dependent genes. In order to elucidate the differential protein profiles between male and female amniocytes, a proteomic approach has been performed in this study. Here, we utilized a proteomics-based approach including 2D-DIGE and MALDI-TOF MS analysis to obtain differentially expressed proteins between male and female amniocytes. After resolving protein samples with 2D-DIGE technique, 45 proteins corresponding to 28 unique proteins were differentially expressed between male and female amninocytes from three independent batches of amniotic fluid. Of all of these unique identified spots, five of them (annexin A1, cathepsin D, cytoskeletal 19, protein disulfide-isomerase, and vimentin) exhibited more than 1.5-fold upregulation or downregulation in at least two independent experiments. Importantly, the identified proteins involved in protein degradation and protein folding display upregulated in male amniocytes, implying the differential regulations of protein degradation and protein folding during sex development. In conclusion, the identified differentially expressed proteins may be employed as potential signatures for the sex development. Moreover, the established proteomic platform might further utilize to discover the potential biomarkers for the prenatal genetic disorders in fetus.

Hemopexin is up-regulated in plasma from type 1 diabetes mellitus patients: Role of glucose-induced ROS.

Type 1 diabetes mellitus (T1DM) is an insulin-dependent metabolic disease in the world and often occurs in children and adolescents. Recent advances in quantitative proteomics offer potential for the discovery of plasma proteins as biomarkers for tracking disease progression and for understanding the molecular mechanisms of diabetes. Comparative proteomic analysis of the plasma proteomes from T1DM cases and healthy donors with lysine- and cysteine-labeling 2D-DIGE combining MALDI-TOF/TOF mass spectrometry revealed that 39 identified T1DM-associated plasma proteins showed significant changes in protein expression including hemopexin, and 41 in thiol reactivity. Further study showed that hemopexin can be induced in numerous cell lines by increasing the glucose concentration in the medium. Interestingly, glucose-induced hemopexin expression can be reduced by reactive oxygen species (ROS) scavengers such as glutathione, implying that hemopexin expression is linked to glucose-induced oxidative stress. In conclusion, the current work has identified potential T1DM biomarkers and one of these, hemopexin, can be modulated by glucose through a ROS-dependent mechanism.

Proteomic and redox-proteomic analysis of berberine-induced cytotoxicity in breast cancer cells.

Berberine is a natural product isolated from herbal plants such as Rhizoma coptidis which has been shown to have anti-neoplastic properties. However, the effects of berberine on the behavior of breast cancers are largely unknown. To determine if berberine might be useful in the treatment of breast cancer and its cytotoxic mechanism, we analyzed the impact of berberine treatment on differential protein expression and redox regulation in human breast cancer cell line MCF-7 using lysine- and cysteine-labeling two-dimensional difference gel electrophoresis (2D-DIGE) combined with mass spectrometry (MS). This study demonstrated that 96 and 22 protein features were significantly changed in protein expression and thiol reactivity, respectively and revealed that berberine-induced cytotoxicity in breast cancer cells involves dysregulation of protein folding, proteolysis, redox regulation, protein trafficking, cell signaling, electron transport, metabolism and centrosomal structure. Our work shows that this combined proteomic strategy provides a rapid method to study the molecular mechanisms of berberine-induced cytotoxicity in breast cancer cells. The identified targets may be useful for further evaluation as potential targets in breast cancer therapy.

Proteomic analysis of gemcitabine-induced drug resistance in pancreatic cancer cells.

Currently, the most effective agent against pancreatic cancer is gemcitabine (GEM), which inhibits tumor growth by interfering with DNA replication and blocking DNA synthesis. However, GEM-induced drug resistance in pancreatic cancer compromises the therapeutic efficacy of GEM. To investigate the molecular mechanisms associated with GEM-induced resistance, 2D-DIGE and MALDI-TOF mass spectrometry were performed to compare the proteomic alterations of a panel of differential GEM-resistant PANC-1 cells with GEM-sensitive pancreatic cells. The proteomic results demonstrated that 33 proteins were differentially expressed between GEM-sensitive and GEM-resistant pancreatic cells. Of these, 22 proteins were shown to be resistance-specific and dose-dependent in the regulation of GEM. Proteomic analysis also revealed that proteins involved in biosynthesis and detoxification are significantly over-expressed in GEM-resistant PANC-1 cells. In contrast, proteins involved in vascular transport, bimolecular decomposition, and calcium-dependent signal regulation are significantly over-expressed in GEM-sensitive PANC-1 cells. Notably, both protein-protein interaction of the identified proteins with bioinformatic analysis and immunoblotting results showed that the GEM-induced pancreatic cell resistance might interplay with tumor suppressor protein p53. Our approach has been shown here to be useful for confidently detecting pancreatic proteins with differential resistance to GEM. Such proteins may be functionally involved in the mechanism of chemotherapy-induced resistance.

Plasma proteomic analysis of the critical limb ischemia markers in diabetic patients with hemodialysis.

Critical limb ischemia (CLI) is a severe obstruction of the arteries resulting from seriously decreased blood flow to the extremities, progressing to the point of pain and even skin ulcers or sores. CLI is associated with a high percentage of limb loss and mortality; however, no reliable biochemical indices are available to monitor the stages of CLI. We developed a strategy involving comparative proteomic analysis to detect CLI associated plasma biomarkers. 2D-DIGE and subsequent MALDI-TOF MS analyses provided 50 differentially expressed plasma proteins (including alkaline phosphatase and haptoglobin), between hemodialytic diabetic patients with and without CLI. Interestingly, more than half of the differentially expressed plasma proteins are associated with inflammatory responses. Our results show that CLI is strongly correlated to inflammation, indicating a strong potential for proteomics analysis in the diagnosis of CLI. To the best of our knowledge, this is the first report presenting a proteomics approach to monitor differentially expressed plasma proteins associated with CLI.

Mitochondrial proteomics analysis of tumorigenic and metastatic breast cancer markers.

Mitochondria are key organelles in mammary cells responsible for several cellular functions including growth, division, and energy metabolism. In this study, mitochondrial proteins were enriched for proteomics analysis with the state-of-the-art two-dimensional differential gel electrophoresis and matrix-assistant laser desorption ionization-time-of-flight mass spectrometry strategy to compare and identify the mitochondrial protein profiling changes between three breast cell lines with different tumorigenicity and metastasis. The proteomics results demonstrate more than 1,500 protein features were resolved from the equal amount pooled from three purified mitochondrial proteins, and 125 differentially expressed spots were identified by their peptide finger print, in which, 33 identified proteins belonged to mitochondrial proteins. Eighteen out of these 33 identified mitochondrial proteins such as SCaMC-1 have not been reported in breast cancer research to our knowledge. Additionally, mitochondrial protein prohibitin has shown to be differentially distributed in mitochondria and in nucleus for normal breast cells and breast cancer cell lines, respectively. To sum up, our approach to identify the mitochondrial proteins in various stages of breast cancer progression and the identified proteins may be further evaluated as potential breast cancer markers in prognosis and therapy.

Trypsin-induced proteome alteration during cell subculture in mammalian cells.

BACKGROUND: It is essential to subculture the cells once cultured cells reach confluence. For this, trypsin is frequently applied to dissociate adhesive cells from the substratum. However, due to the proteolytic activity of trypsin, cell surface proteins are often cleaved, which leads to dysregulation of the cell functions. METHODS: In this study, a triplicate 2D-DIGE strategy has been performed to monitor trypsin-induced proteome alterations. The differentially expressed spots were identified by MALDI-TOF MS and validated by immunoblotting. RESULTS: 36 proteins are found to be differentially expressed in cells treated with trypsin, and proteins that are known to regulate cell metabolism, growth regulation, mitochondrial electron transportation and cell adhesion are down-regulated and proteins that regulate cell apoptosis are up-regulated after trypsin treatment. Further study shows that bcl-2 is down-regulated, p53 and p21 are both up-regulated after trypsinization. CONCLUSIONS: In summary, this is the first report that uses the proteomic approach to thoroughly study trypsin-induced cell physiological changes and provides researchers in carrying out their experimental design.

Secretomic and proteomic analysis of potential breast cancer markers by two-dimensional differential gel electrophoresis.

The transformation of a normal cell into a cancer cell has been correlated with alterations in gene regulation and protein expression. To identify altered proteins and link them to the tumorigenesis of breast cancer, we have distinguished normal breast cells (MCF-10A) from noninvasive breast cancer cells (MCF-7) and invasive breast cancer cells (MB-MDA-231) to identify potential breast cancer markers in transformed breast cells. Using the 2D-DIGE and MALDI-TOF MS techniques, we quantified and identified differentially expressed extracellular secreted proteins and total cellular proteins across MCF-7, MB-MDA-231 and MCF-10A. The proteomic analysis of the secreted proteins identified 50 unique differentially expressed proteins from three different media. In addition, 133 unique differentially expressed proteins from total cellular proteins were also identified. Note that 14 of the secreted proteins and 51 of the total cellular proteins have not been previously reported in breast cancer research. Some of these unreported proteins have been examined in other breast cancer cell lines and have shown positive correlations with 2D-DIGE data. In summary, this study identifies numerous putative breast cancer markers from various stages of breast cancer. The results of this study may aid in developing proteins identified as useful diagnostic and therapeutic candidates in research on cancer and proteomics.