ChipFilter: Microfluidic-Based Comprehensive Sample Preparation Methodology for Microbial Consortia.

The metaproteomic approach is an attractive way to describe a microbiome at the functional level, allowing the identification and quantification of proteins across a broad dynamic range as well as the detection of post-translational modifications. However, it remains relatively underutilized, mainly due to technical challenges that should be addressed, including the complexity of extracting proteins from heterogeneous microbial communities. Here, we show that a ChipFilter microfluidic device coupled to a liquid chromatography tandem mass spectrometry (LC-MS/MS) setup can be successfully used for the identification of microbial proteins. Using cultures of Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae, we have shown that it is possible to directly lyse the cells and digest the proteins in the ChipFilter to allow the identification of a higher number of proteins and peptides than that by standard protocols, even at low cell density. The peptides produced are overall longer after ChipFilter digestion but show no change in their degree of hydrophobicity. Analysis of a more complex mixture of 17 species from the gut microbiome showed that the ChipFilter preparation was able to identify and estimate the amounts of 16 of these species. These results show that ChipFilter can be used for the proteomic study of microbiomes, particularly in the case of a low volume or cell density. The mass spectrometry data have been deposited on the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD039581.

Identification of dysregulation of sphingolipids in retinoblastoma using liquid chromatography-mass spectrometry.

Retinoblastoma (RB) is a rare ocular cancer seen in children that counts for approximately 3% of all childhood cancers. It is found that mutation in RB1, a tumour Suppressor Gene on chromosome 13 as the cause of malignancy. Retinoblastoma protein is the target for ceramide to cause apoptosis. We studied lipidomics of two RB cell lines, one aggressive cell line (NCC-RbC-51) derived from a metastatic site and one non aggressive cell line (WERI-Rb1) in comparison with a control cell line (MIO-M1). Lipid profiles of all the cell lines were studied using high resolution mass spectrometer coupled to high performance liquid chromatography. Data acquired from all the three cell lines in positive mode were analyzed to identify differentially expressed metabolites. Several phospholipids and lysophospholipids were found to be dysregulated. We observed upregulation of hexosyl ceramides, and down regulation of dihydroceramides and higher order sphingoglycolipids hinting at a hindered sphingolipid biosynthesis. The results obtained from liquid chromatography-mass spectrometry are validated by using qPCR and it was observed that genes involved in ceramide biosynthesis pathway are getting down regulated.

Metabolite systems profiling identifies exploitable weaknesses in retinoblastoma.

Retinoblastoma (RB) is a childhood eye cancer. Currently, chemotherapy, local therapy, and enucleation are the main ways in which these tumors are managed. The present work is the first study that uses constraint-based reconstruction and analysis approaches to identify and explain RB-specific survival strategies, which are RB tumor specific. Importantly, our model-specific secretion profile is also found in RB1-depleted human retinal cells in vitro and suggests that novel biomarkers involved in lipid metabolism may be important. Finally, RB-specific synthetic lethals have been predicted as lipid and nucleoside transport proteins that can aid in novel drug target development.