Evaluation of freely available software tools for untargeted quantification of 13C isotopic enrichment in cellular metabolome from HR-LC/MS data.

13C Metabolic Flux Analysis (13C-MFA) involves the quantification of isotopic enrichment in cellular metabolites and fitting the resultant data to the metabolic network model of the organism. Coverage and resolution of the resultant flux map depends on the total number of metabolites and fragments in which 13C enrichment can be quantified accurately. Experimental techniques for tracking 13C enrichment are evolving rapidly and large volumes of data are now routinely generated through the use of Liquid Chromatography coupled with High-Resolution Mass Spectrometry (HR-LC/MS). Therefore, the current manuscript is focused on the challenges in high-throughput analyses of such large datasets. Current 13C-MFA studies often have to rely on the targeted quantification of a small subset of metabolites, thereby leaving a large fraction of the data unexplored. A number of public domain software tools have been reported in recent years for the untargeted quantitation of isotopic enrichment. However, the suitability of their application across diverse datasets has not been investigated. Here, we test the software tools X13CMS, DynaMet, geoRge, and HiResTEC with three diverse datasets. The tools provided a global, untargeted view of 13C enrichment in metabolites in all three datasets and a much-needed automation in data analysis. Some inconsistencies were observed in results obtained from the different tools, which could be partially ascribed to the lack of baseline separation and potential mass conflicts. After removing the false positives manually, isotopic enrichment could be quantified reliably in a large repertoire of metabolites. Of the software tools explored, geoRge and HiResTEC consistently performed well for the untargeted analysis of all datasets tested.

Mass spectrometry based identification of galectin-3 interacting proteins potentially involved in lung melanoma metastasis.

Adhesive interactions between molecules on tumor cells and those on target organs play a key role in organ specific metastasis. Poly-N-acetyl-lactosamine (polyLacNAc) substituted N-oligosaccharides on melanoma cell surface glycoproteins promote lung specific metastasis via galectin-3 by facilitating their arrest and extravasation. This study reports the identification and characterization of galectin-3 interacting proteins using a combination of galectin-3 sepharose affinity and leucoagglutinating phytohemagglutinin (L-PHA) columns. A total of 83 proteins were identified as galectin-3 interacting glycoproteins, of which 35 were constituents of the L-PHA bound fraction, suggesting that these proteins carry polyLacNAc substituted ß1,6 branched N-glycans. The identities of some of these proteins, like LAMP-1, LAMP-3, basigin, embigin, and α5 and ß1 Integrin, have been confirmed by western blotting, and functional relevance with respect to metastatic properties has been established.

Dynamic phosphorylation of RelA on Ser42 and Ser45 in response to TNFα stimulation regulates DNA binding and transcription.

The NF-κB signalling module controls transcription through a network of protein kinases such as the IKKs, as well as inhibitory proteins (IκBs) and transcription factors including RelA/p65. Phosphorylation of the NF-κB subunits is critical for dictating system dynamics. Using both non-targeted discovery and quantitative selected reaction monitoring-targeted proteomics, we show that the cytokine TNFα induces dynamic multisite phosphorylation of RelA at a number of previously unidentified residues. Putative roles for many of these phosphorylation sites on RelA were predicted by modelling of various crystal structures. Stoichiometry of phosphorylation determination of Ser45 and Ser42 revealed preferential early phosphorylation of Ser45 in response to TNFα. Quantitative analyses subsequently confirmed differential roles for pSer42 and pSer45 in promoter-specific DNA binding and a role for both of these phosphosites in regulating transcription from the IL-6 promoter. These temporal dynamics suggest that RelA-mediated transcription is likely to be controlled by functionally distinct NF-κB proteoforms carrying different combinations of modifications, rather than a simple 'one modification, one effect' system.

Extracellular galectin-3 induces MMP9 expression by activating p38 MAPK pathway via lysosome-associated membrane protein-1 (LAMP1).

Matrix metalloproteinases (MMPs) play a key role in matrix remodelling and thus invasion and metastasis. Extracellular galectin-3 has been shown to induce MMP9 secretion. Here, we demonstrate that galectin-3 induces MMP9 at transcript level and it is dependent on the surface levels of poly-N-acetyllactosamine (polyLacNAc). By employing signalling pathway inhibitors, MMP9 expression was shown to be induced via p38 MAP-kinase pathway. Using clones of melanoma cells expressing shRNAs to lysosome-associated membrane protein-1 (LAMP1), a major carrier of polyLacNAc, surface LAMP1 was demonstrated to serve as one of the key mediators of galectin-3-induced MMP9 expression via p38 MAPK pathway.

Galectin-3 expressed on different lung compartments promotes organ specific metastasis by facilitating arrest, extravasation and organ colonization via high affinity ligands on melanoma cells.

Interactions between molecules on the surface of tumor cells and those on the target organ endothelium play an important role in their arrest in an organ. Galectin-3 on the lung endothelium and high affinity ligands poly-N-acetyllactosamine (polyLacNAc) on N-oligosaccharides on melanoma cells facilitate such interactions. However, to extravasate and colonize an organ the cells must stabilize these interactions by spreading to retract endothelium, degrade exposed basement membrane (BM) and move into parenchyma and proliferate. Here, we show that galectin-3 is expressed on all the major compartments of the lungs and participates in not just promoting adhesion but also in spreading. We for the first time demonstrate that both soluble and immobilized galectin-3 induce secretion of MMP-9 required to breach vascular BM. Further, we show that immobilized galectin-3 is used as traction for the movement of cells. Downregulation of galactosyltransferases-I and -V resulted in significant loss in expression of polyLacNAc and thus reduced binding of galectin-3. This was accompanied with a loss in adhesion, spreading, MMP-9 secretion and motility of the cells on galectin-3 and thus their metastasis to lungs. Metastasis could also be inhibited by blocking surface polyLacNAc by pre-incubating cells with truncated galectin-3 (which lacked oligomerization domain) or by feeding mice with modified citrus pectin in drinking water. Overall, these results unequivocally show that polyLacNAc on melanoma cells and galectin-3 on the lungs play a critical role in arrest and extravasation of cells in the lungs and strategies that target these interactions inhibit lung metastasis.