We apply stable isotope dilution methods to quantify metabolites/lipids from biological matrices like dried blood spots, serum, urine, tissue and cell extracts. Our methods can detect sub picogram levels of metabolites. In case of amines, we use aminoquinoline carbamate derivatization to enhance the sensitivity from biological fluids. We use the advanced technologies using Altis TSQ instrument for all our targeted analysis. Currently we can quantify 70 amines (amino acids, neurotransmitters and amino acid derivatives), 30 organic acids, 40 carnitines and various classes of lipids like ceramides, dihydroceramides, glucosyl ceramides, phosphatidyl cholines, phosphatidyl ethanolamine, phosphatidyl serines and free fatty acids. Similarly, we develop LC-MS/SRM based methods for other chemical classes of metabolite present in biological samples.
We use the untargeted approach in a discovery phase to identify differently expressed metabolites between two sample sets (e.g. healthy Vs patient). We inject extracted metabolites/lipids through HPLC connected to a high-resolution mass spectrometer. We apply different chromatographic approaches to cover all kinds of metabolites/lipids in two different polarities (positive and negative). A software processes the data from LC-MS for retention time alignment, statistical analysis and for the identification of differently abundant molecular features using their publicly available accurate mass information and or by their fragmentation spectra. Different kinds of tools like PCA, PLS-DA, OPLS-DA can help in categorizing the sample based on metabolites. This approach is useful to identify disease specific signatures or biomarkers in patient samples.
Rare diseases are mostly due to a mutation in a gene affecting a small percentage of the population in early life, approximately about 30% of children. It is very important to identify the disease in its early state to prevent permanent damage. Despite of genetic mutation, sometimes clinical presentation may change. Metabolomic studies on patient and control samples will help in understanding the genotype-phenotype difference and to identify disease specific markers. Currently, we use different tools to identify different kinds of metabolites. Our goal is to provide a one-stop solution to identify a wide variety of molecules by a single technique. We develop methods by using both targeted and untargeted approaches to look at various metabolic disorders like aminoacidemia, organic aciduria, fatty acid oxidation disorders, lipid storage disorders, mucopolysaccharidosis and other lysosomal storage disorders.
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 are studying ceramide pathway in two RB cell lines, one aggressive cell line (NCC RbC 51) and one non aggressive cell line (WERI) in comparison with a control cell line (MIOM-1). We observed a severe compromise in sphingolipid pathway in the RB cell lines. Sphingoglycolipids are found to be upregulated in RB cell lines.