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.
Glycoproteomics is a branch of proteomics that is focused on
identifying, cataloguing and characterizing proteins which have been modified
by addition of carbohydrate moieties during the process of post-translational
modifications. We have optimized different enrichment strategies to detect the
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 strong anion exchange chromatography, mixed anion exchange
chromatography, size exclusion chromatography and lectin affinity chromatography.
We have explored N-linked glycans. We have profiled proteoglycans especially
chondroitin sulfate linked proteoglycans and we are currently exploring methods
to characterize other proteoglycans using high resolution mass spectrometry.
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
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.
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.