Molecular Biology Lab
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Quantitative proteomics

We use quantitative proteomics approaches for the identification of candidate biomarkers in human diseases and infectious microorganisms. We have pioneered the use of in vivo labeling strategies such as SILAC (stable isotope labeling with amino acids in cell culture), a technology that is currently being used by numerous groups around the world.  We are also experts in other quantitative labeling methodologies such as 18O labeling and iTRAQ –in fact, we were early adopters of the 8-plex labeling reagents, which will be the mainstay of many of the studies to be carried out under this initiative.  We characterized proteome of various cancers including breast, pancreatic, hepatocellular, lung and billiary cancers. We are adopting same technologies to identify molecular profiling of esophageal squamous cell, gastric and gall bladder carcinoma.  Similarly, we are analyzing proteome of other diseases such as chronic meningitis, neurological stroke, temporal lobe epilepsy and rabies.



We are investigating phosphoproteomic profiling associated with various cancers and other diseases. We are also profiling phosphotyrosines in various human tissues, cancers, other diseases and several microbes of clinical and industrial importance. Hyperphosphorylated and hypophosphorylated molecules associated with a particular disease, virulence or drug resistance collectively may provide information about activation or down-regulation of associated signaling pathways or molecular mechanisms. We combine the advantage of the SILAC or iTRAQ method to obtain quantitative data with TiO2 and anti- phosphopeptide antibody based enrichment of phosphopeptides.  We were also the first to apply a novel mass spectrometric technology, Electron Transfer Dissociation (ETD), for global phosphoproteomic analysis. ETD is a preferred method of fragmentation in phosphoproteomic studies as it preserves phosphate moieties on serine and threonine residues that are otherwise quite labile in conventional collision induced dissociation.


Proteomics of Body Fluids

The main aim of this research is to generate and quantitatively identify the complete proteomic profiles of every protein expressed in various human body fluids using our state-of-the-art and highly sensitive mass spectroscopy-based proteomic techniques. We summarize some of the strategies currently being used for global identification and quantification of body fluid proteins with applications for a wide variety of human diseases. Our findings reveal novel protein species in both healthy and disease states that were not previously identified. While it is reasonable to expect that some of the same proteins are found in multiple body fluids, the primary objective of this research will be to identify a definable subset of proteins that were unique to or diagnostic for a single body fluid. The study will evaluate the entire complement of proteins in each proteome for potential utility as bodily fluid specific biomarkers. Consequently we hope that some of these biomarkers can be further characterized for use in highly sensitive diagnostic tests or for evaluating therapeutic response. With the advances made in proteomics technologies, the impact of this analysis in the search for clinically relevant disease biomarkers would be realized in the future.



We are using glycoproteome enrichment techniques in order to detect usually less abundant glycosylated proteins that are more relevant from the point of view of biomarker development. We have substantial experience in enrichment of glycoproteins using lectin affinity chromatography. The enrichment is carried out using a mixture of three lectins - concanavalin A, wheat germ agglutinin and jacalin- that are immobilized on a matrix.  Use of 3 lectins provides a broader coverage of glycoproteins because of their broader binding profile and can provide up to 1,000 fold enrichment of molecules in a single step. For identification of N-linked glycopeptides, the extracted tryptic peptides are treated with PNGaseF.  This digestion step removes the N-linked glycan and is recognized by a deamidation at the glycan linked asparagine during cleavage. This converts asparagine to aspartic acid increasing the mass by 1Da that could be very easily identified by mass spectrometry.


Cell line secretome

The secretome of cells and tissues reflects a wide range of pathological conditions and represents as a good source of biomarkers. The identification of secreted proteins is usually hard as it is hardly accessible by direct proteome analysis because these proteins are often masked by high amounts of proteins not secreted by the cells under investigation. Therefore, we study the secretome from patient derived cell lines as that can give a better idea of the secretome abundance of protein in tumor as oppose to normal. We have successfully used SILAC technology and cell line secretome approach to investigate secreted biomarkers of pancreatic and esophageal cancers.



Proteogenomics is the annotation of genomes using proteomics data. Mass spectrometry derived data can be searched against genome which will provide the evidence for protein coding potential.  Successful outcome of such analyses include identification of novel genes, novel exons, novel initiation codons, exon extensions, intronic genes and cSNPs. We are carrying out proteogenomic analysis of Anopheles gambiae, A. stephensi, Mycobacterium tuberculosis and Candida glabrata. Using proteogenomic approach, we have corrected existing gene models for 199 genes, identified 35 novel genes, and assigned several novel translational start sites in the genome of A. gambiae.



We are adopting proteomic, phosphoproteomic, glycoproteomic approaches to identify molecular markers for several neurological diseases such as chronic meningitis, rabies, temporal lobe epilepsy and neurological stroke. 


Plant proteomics

Mangifera indica, national fruit of India, is one of important fruit crop. Although extensive investigations have been carried out in raising improved varieties of mango cultivars, molecular characterization remains unexplored. Towards this end, we are carrying out proteomic profiling of mango to provide a platform for future investigations on molecular level to study crop improvement, disease resistance and pest resistance. This is particularly challenging because genome of mango hasn’t been sequenced yet. Therefore, we are depending on homology based search and de novo sequencing to annotate mass spectrometry derived data.




We are using quantitative proteomics, phosphoproteomics, glycoproteomics and cell line secretome pipelines to study the response of human cells to toxic pollutants. We are analyzing differential proteomics of Hep G2 cells exposed to lead and arsenic with respect to untreated controls. The differentially regulated proteins may serve as candidate biomarker for diagnosing lead or arsenic toxicity and for deciding treatment options. Quantitative profiling of phosphoproteome in the cell lines exposed to lead using high resolution mass spectrometry may help us to identify signaling pathways activated or inhibited upon lead toxicity.




India is rich in terms of cultural knowledge associated with traditional medicine, mainly comprised of usage of herbs. We are adopting the ethnobotanical principles to test their effect on diseases in particular or on human physiology in general using quantitative mass spectrometry approaches. For example, curcumin, a product of Curcuma longa, has been identified as one of the major natural anticancer agents exerting anti-neoplastic activity in various types of cancers tested. Using diverse proteomic platforms we specifically identify proteins that are differentially regulated in breast cancer upon curcumin treatment. We are investigating differentially induced phosphorylation of proteins in breast cancer cells upon curcumin treatment. We can also identify molecules and signaling pathways that are associated with anti-tumor activity of curcumin by means of data generated by global proteome and phosphoproteome profiles. It is possible that a better understanding of the mechanistic details of curcumin action in breast cancer will lead to design of more targeted strategies to combat breast cancer.