High-Performance Graphene-Titania Platform for Detection of Phosphopeptides in Cancer Cells

Phosphopeptides play a crucial role in many biological processes and constitute some of the most powerful biomarkers in disease detection. However they are often present in very low concentration, which makes their detection highly challenging. Here, we demonstrate the use of a solution-dispersible graphene-titania platform for the selective extraction of phosphopeptides from peptide mixtures. This is followed by direct analysis by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). The efficient charge and energy exchange between graphene and TiO₂ during laser irradiation in SELDI-TOF MS promotes the soft ionization of analytes and affords a detection limit in the attomole range, which is 10²–10⁵ more sensitive than conventional platforms. The graphene-titania platform can also be used for detecting phosphopeptides in cancer cells (HeLa cells), where it shows high specificity (94%). An expanded library of 967 unique phosphopeptides is detected using significantly reduced loading of extraction matrixes compared to conventional TiO₂ bead-based assays

High-Performance Graphene-Titania Platform for Detection of Phosphopeptides in Cancer Cells

Phosphopeptides play a crucial role in many biological processes and constitute some of the most powerful biomarkers in disease detection. However they are often present in very low concentration, which makes their detection highly challenging. Here, we demonstrate the use of a solution-dispersible graphene-titania platform for the selective extraction of phosphopeptides from peptide mixtures. This is followed by direct analysis by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). The efficient charge and energy exchange between graphene and TiO₂ during laser irradiation in SELDI-TOF MS promotes the soft ionization of analytes and affords a detection limit in the attomole range, which is 10²–10⁵ more sensitive than conventional platforms. The graphene-titania platform can also be used for detecting phosphopeptides in cancer cells (HeLa cells), where it shows high specificity (94%). An expanded library of 967 unique phosphopeptides is detected using significantly reduced loading of extraction matrixes compared to conventional TiO₂ bead-based assays

Proteomic Analysis of the Oil Palm Fruit Mesocarp Reveals Elevated Oxidative Phosphorylation Activity is Critical for Increased Storage Oil Production

Palm oil is a highly versatile commodity with wide applications in the food, cosmetics, and biofuel industries. Storage oil in the oil palm mesocarp can make up a remarkable 80% of its dry mass, making it the oil crop with the richest oil content in the world. As such, there has been an ongoing interest in understanding the mechanism of oil production in oil palm fruits. To identify the proteome changes during oil palm fruit maturation and factors affecting oil yield in oil palm fruits, we examined the proteomic profiles of oil palm mesocarps at four developing stages – 12, 16, 18, and 22 weeks after pollination – by 8-plex iTRAQ labeling coupled to 2D-LC and MALDI-TOF/TOF MS. It was found that proteins from several important metabolic processes, including starch and sucrose metabolism, glycolysis, pentose phosphate shunt, fatty acid biosynthesis, and oxidative phosphorylation, were differentially expressed in a concerted manner. These increases led to an increase in carbon flux and a diversion of resources such as ATP and NADH that are required for lipid biosynthesis. The temporal proteome profiles between the high-oil-yielding (HY) and low-oil-yielding (LY) fruits also showed significant differences in the levels of proteins involved in the regulation of the TCA cycle and oxidative phosphorylation. In particular, the expression level of the β subunit of the ATP synthase complex (complex IV of the electron transport chain) was found to be increased during fruit maturation in HY but decreased in the LY during the fruit maturation. These results suggested that increased energy supply is necessary for augmented oil yield in the HY oil palm trees

Proteomic Analysis of the Oil Palm Fruit Mesocarp Reveals Elevated Oxidative Phosphorylation Activity is Critical for Increased Storage Oil Production

Palm oil is a highly versatile commodity with wide applications in the food, cosmetics, and biofuel industries. Storage oil in the oil palm mesocarp can make up a remarkable 80% of its dry mass, making it the oil crop with the richest oil content in the world. As such, there has been an ongoing interest in understanding the mechanism of oil production in oil palm fruits. To identify the proteome changes during oil palm fruit maturation and factors affecting oil yield in oil palm fruits, we examined the proteomic profiles of oil palm mesocarps at four developing stages – 12, 16, 18, and 22 weeks after pollination – by 8-plex iTRAQ labeling coupled to 2D-LC and MALDI-TOF/TOF MS. It was found that proteins from several important metabolic processes, including starch and sucrose metabolism, glycolysis, pentose phosphate shunt, fatty acid biosynthesis, and oxidative phosphorylation, were differentially expressed in a concerted manner. These increases led to an increase in carbon flux and a diversion of resources such as ATP and NADH that are required for lipid biosynthesis. The temporal proteome profiles between the high-oil-yielding (HY) and low-oil-yielding (LY) fruits also showed significant differences in the levels of proteins involved in the regulation of the TCA cycle and oxidative phosphorylation. In particular, the expression level of the β subunit of the ATP synthase complex (complex IV of the electron transport chain) was found to be increased during fruit maturation in HY but decreased in the LY during the fruit maturation. These results suggested that increased energy supply is necessary for augmented oil yield in the HY oil palm trees

Proteomic Analysis of the Oil Palm Fruit Mesocarp Reveals Elevated Oxidative Phosphorylation Activity is Critical for Increased Storage Oil Production

Palm oil is a highly versatile commodity with wide applications in the food, cosmetics, and biofuel industries. Storage oil in the oil palm mesocarp can make up a remarkable 80% of its dry mass, making it the oil crop with the richest oil content in the world. As such, there has been an ongoing interest in understanding the mechanism of oil production in oil palm fruits. To identify the proteome changes during oil palm fruit maturation and factors affecting oil yield in oil palm fruits, we examined the proteomic profiles of oil palm mesocarps at four developing stages – 12, 16, 18, and 22 weeks after pollination – by 8-plex iTRAQ labeling coupled to 2D-LC and MALDI-TOF/TOF MS. It was found that proteins from several important metabolic processes, including starch and sucrose metabolism, glycolysis, pentose phosphate shunt, fatty acid biosynthesis, and oxidative phosphorylation, were differentially expressed in a concerted manner. These increases led to an increase in carbon flux and a diversion of resources such as ATP and NADH that are required for lipid biosynthesis. The temporal proteome profiles between the high-oil-yielding (HY) and low-oil-yielding (LY) fruits also showed significant differences in the levels of proteins involved in the regulation of the TCA cycle and oxidative phosphorylation. In particular, the expression level of the β subunit of the ATP synthase complex (complex IV of the electron transport chain) was found to be increased during fruit maturation in HY but decreased in the LY during the fruit maturation. These results suggested that increased energy supply is necessary for augmented oil yield in the HY oil palm trees

Mining the Gastric Cancer Secretome: Identification of GRN as a Potential Diagnostic Marker for Early Gastric Cancer

Gastric cancer is the second leading cause of cancer deaths worldwide, and currently, there are no clinically relevant biomarkers for gastric cancer diagnosis or prognosis. In this study, we applied a 2D-LC-MS/MS based approach, in combination with iTRAQ labeling, to study the secretomes of the gastric cancer cell lines AGS and MKN7. By performing a comparative analysis between the conditioned media and the whole cell lysates, our workflow allowed us to differentiate the <i>bona fide</i> secreted proteins from the intracellular contaminants within the conditioned media. Ninety proteins were found to have higher abundance in the conditioned media as compared to the whole cell lysates of AGS and MKN7 cells. Using a signal peptide and nonclassical secretion prediction tool and an online exosome database, we demonstrated that up to 92.2% of these 90 proteins can be exported out of the cells by classical or nonclassical secretory pathways. We then performed quantitative comparisons of the secretomes between AGS and MKN7, identifying 43 differentially expressed secreted proteins. Among them, GRN was found to be frequently expressed in gastric tumor tissues, but not in normal gastric epithelia by immunohistochemistry. Sandwich ELISA assay also showed elevation of serum GRN levels in gastric cancer patients, particularly those with early gastric cancer. Receiver operating characteristic (ROC) curves analysis confirmed that serum GRN can provide diagnostic discriminations for gastric cancer patient

Mining the Gastric Cancer Secretome: Identification of GRN as a Potential Diagnostic Marker for Early Gastric Cancer

Gastric cancer is the second leading cause of cancer deaths worldwide, and currently, there are no clinically relevant biomarkers for gastric cancer diagnosis or prognosis. In this study, we applied a 2D-LC-MS/MS based approach, in combination with iTRAQ labeling, to study the secretomes of the gastric cancer cell lines AGS and MKN7. By performing a comparative analysis between the conditioned media and the whole cell lysates, our workflow allowed us to differentiate the <i>bona fide</i> secreted proteins from the intracellular contaminants within the conditioned media. Ninety proteins were found to have higher abundance in the conditioned media as compared to the whole cell lysates of AGS and MKN7 cells. Using a signal peptide and nonclassical secretion prediction tool and an online exosome database, we demonstrated that up to 92.2% of these 90 proteins can be exported out of the cells by classical or nonclassical secretory pathways. We then performed quantitative comparisons of the secretomes between AGS and MKN7, identifying 43 differentially expressed secreted proteins. Among them, GRN was found to be frequently expressed in gastric tumor tissues, but not in normal gastric epithelia by immunohistochemistry. Sandwich ELISA assay also showed elevation of serum GRN levels in gastric cancer patients, particularly those with early gastric cancer. Receiver operating characteristic (ROC) curves analysis confirmed that serum GRN can provide diagnostic discriminations for gastric cancer patient

Mining the Gastric Cancer Secretome: Identification of GRN as a Potential Diagnostic Marker for Early Gastric Cancer

Gastric cancer is the second leading cause of cancer deaths worldwide, and currently, there are no clinically relevant biomarkers for gastric cancer diagnosis or prognosis. In this study, we applied a 2D-LC-MS/MS based approach, in combination with iTRAQ labeling, to study the secretomes of the gastric cancer cell lines AGS and MKN7. By performing a comparative analysis between the conditioned media and the whole cell lysates, our workflow allowed us to differentiate the <i>bona fide</i> secreted proteins from the intracellular contaminants within the conditioned media. Ninety proteins were found to have higher abundance in the conditioned media as compared to the whole cell lysates of AGS and MKN7 cells. Using a signal peptide and nonclassical secretion prediction tool and an online exosome database, we demonstrated that up to 92.2% of these 90 proteins can be exported out of the cells by classical or nonclassical secretory pathways. We then performed quantitative comparisons of the secretomes between AGS and MKN7, identifying 43 differentially expressed secreted proteins. Among them, GRN was found to be frequently expressed in gastric tumor tissues, but not in normal gastric epithelia by immunohistochemistry. Sandwich ELISA assay also showed elevation of serum GRN levels in gastric cancer patients, particularly those with early gastric cancer. Receiver operating characteristic (ROC) curves analysis confirmed that serum GRN can provide diagnostic discriminations for gastric cancer patient

Proteomic Analysis of the Oil Palm Fruit Mesocarp Reveals Elevated Oxidative Phosphorylation Activity is Critical for Increased Storage Oil Production

Palm oil is a highly versatile commodity with wide applications in the food, cosmetics, and biofuel industries. Storage oil in the oil palm mesocarp can make up a remarkable 80% of its dry mass, making it the oil crop with the richest oil content in the world. As such, there has been an ongoing interest in understanding the mechanism of oil production in oil palm fruits. To identify the proteome changes during oil palm fruit maturation and factors affecting oil yield in oil palm fruits, we examined the proteomic profiles of oil palm mesocarps at four developing stages – 12, 16, 18, and 22 weeks after pollination – by 8-plex iTRAQ labeling coupled to 2D-LC and MALDI-TOF/TOF MS. It was found that proteins from several important metabolic processes, including starch and sucrose metabolism, glycolysis, pentose phosphate shunt, fatty acid biosynthesis, and oxidative phosphorylation, were differentially expressed in a concerted manner. These increases led to an increase in carbon flux and a diversion of resources such as ATP and NADH that are required for lipid biosynthesis. The temporal proteome profiles between the high-oil-yielding (HY) and low-oil-yielding (LY) fruits also showed significant differences in the levels of proteins involved in the regulation of the TCA cycle and oxidative phosphorylation. In particular, the expression level of the β subunit of the ATP synthase complex (complex IV of the electron transport chain) was found to be increased during fruit maturation in HY but decreased in the LY during the fruit maturation. These results suggested that increased energy supply is necessary for augmented oil yield in the HY oil palm trees

Identification and Functional Validation of Caldesmon as a Potential Gastric Cancer Metastasis-associated Protein

In this study, we aim to identify biomarkers for gastric cancer metastasis using a quantitative proteomics approach. The proteins extracted from a panel of 4 gastric cancer cell lines, two derived from primary cancer (AGS, FU97) and two from lymph node metastasis (AZ521, MKN7), were labeled with iTRAQ (8-plex) reagents and analyzed by 2D-LC–MALDI-TOF/TOF MS. In total, 641 proteins were identified with at least a 95% confidence. Using cutoff values of >1.5 and <0.67, 19 proteins were found to be up-regulated and 34 were down-regulated in the metastatic versus primary gastric cancer cell lines respectively. Several of these dysregulated proteins, including caldesmon, were verified using Western blotting. It was found that caldesmon expression was decreased in the two metastasis-derived cell lines, and this was confirmed by further analysis of 7 gastric cancer cell lines. Furthermore, immunohistochemical staining of 9 pairs of primary gastric cancer and the matched lymph node metastasis tissue also corroborated this observation. Finally, knockdown of caldesmon using siRNA in AGS and FU97 gastric cancer cells resulted in an increase in cell migration and invasion, while the overexpression of caldesmon in AZ521 cells led to a decrease in cell migration and invasion. This study has thus established the potential role of caldesmon in gastric cancer metastasis, and further functional studies are underway to delineate the underlying mechanism of action of this protein