Molecular Biology Lab
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Biomarkers in Serum

Blood is the most easily accessible compartment for diagnostic purposes than tissue, identifying biomarkers that are more likely to be detectable in the serum is beneficial over other types of biomarkers. We have experience working with various cancer tissues, serum and other body fluids and we fully understand the technical challenges one faces while analyzing serum specimens for the purpose of identifying low abundance proteins, which is what we expect most of the potential biomarkers to be. As membrane proteins and glycoproteins represent most likely candidates that are shed into the circulation, analysis of these sub-proteomes provides an attractive option for biomarker discovery. Albumin and immunoglobulins that are more abundant proteins in serum interfere with the identification and characterization of low abundant proteins by limiting the dynamic range of identification by mass spectrometry. We routinely use the multiple antigen removal system columns from Agilent Technologies (Human-14) for the depletion of the top 14 high abundance proteins in human serum followed by RP-HPLC using the mRP-C18 column at an elevated temperature. We have established a robust pipeline for the removal of such proteins in serum of patients with pancreatic, esophageal, gastric and gall bladder cancers.

In addition to this, investigating the secretomes of tumor cell lines has evolved as a reasonable alternative approach for discovery of potential biomarkers detectable in serum or blood. Consequently, using a combined strategy of secretome profiling coupled to quantitative mass spectrometry, proteins enriched in the cell supernatants were analyzed for early detection of cancer-specific biomarkers.


Biomarkers in Urine

The ease and non-invasive nature of urine collection combined with the fact that its composition is far less complex compared to serum and blood make it an attractive biological sample for clinical diagnosis. The proteins in urine can be used to screen for many diseases such as cancers and neurodegenerative disorders and serve as an excellent bodily fluid for identification of novel biomarkers for disease. We carried out a comprehensive analysis of human urinary proteome from healthy individuals using high resolution Fourier transform mass spectrometry. We identified 1,823 proteins with less than 1% false discovery rate, of which 671 proteins have not been previously reported in human urine. This dataset should serve as a comprehensive reference list for future studies aimed at identification and characterization of urinary biomarkers for various diseases.


Biomarkers in Saliva

Saliva is the most easily accessible fluid for diagnostic purposes and might offer a non-invasive way to test for diseases and surveillance of oral health. Oral cancer survival rates increase significantly when they are detected and treated early. However, clinicians now lack tests which can reliably distinguish between the different stages of malignancy of oral cancers. Comprehensive analysis and identification of the proteomic content in human saliva is a necessary first step to test for proteins whose abundances can distinguish these lesion types. We have standardized the techniques for depletion of high abundant proteins like amylase and albumin, to investigate the low abundant proteins in saliva. We are currently performing differential proteomics using iTRAQ labeling and high resolution mass spectrometry techniques to explore the presence of protein biomarkers with respect to different stages of oral cancers and to evaluate their potential for diagnostics.


Biomarkers in Synovial Fluid

Arthritis, commonly known as joint disease is further classified into inflammatory and degenerative types and is highly prevalent in India. A key factor in the pathology of joint disease is the synovial fluid that is released by the synovial membrane in the knee joints and normally acts as lubricant and provides nutrients to the cells and tissues of the joints. In the pathogenic site like the knee joint, the fluid accumulation exceeds with severity of the disease and this fluid was collected from knee aspirations of arthritic patients. To identify the proteomic fingerprints of patients suffering from various types of arthritic disorders like rheumatoid arthritis (RA) and spondyloarthropathies (SpA) we carried out differential studies and global profiling of synovial fluid proteins. This fluid is of complex nature and mainly constitutes many highly abundant proteins like albumin and immunoglobulins. Prior to proteomic analysis, the samples are immunodepleted to reduce the complexity and enrich the low abundance protein fractions. We have implemented two different approaches, the lectin affinity based glycoprotein enrichment and the MARS14 depletion of highly abundant proteins for this purpose. Using the LTQ Orbitrap Velos, we have identified proteins which play significant roles in the pathogenesis of arthritis along with novel ones that might further help in biomarker discovery in the field of rheumatology.


Biomarkers in Colostrum and Breast Milk

The importance of colostrum and breast milk for the healthy growth of the infant and protecting them from infection has been well established. They not only serve as a rich source of nutrients but also contain anti-infective factors conferring essential immunity to the newborn. In addition, they also contain certain growth factors, metabolic enzymes and transport proteins which are required for proper growth and development of the infants. A comprehensive analysis of colostrum or milk proteome would help us to better understand their nutritional and physiological implications to the newborn. This may also contribute to developing effective infant formulas similar in protein composition to human milk. Towards this, we have employed both qualitative and quantitative approaches to profile proteins in human colostrum and milk and are in the process of studying the temporal changes associated with colostrum and milk proteome.


Biomarkers in Bile

Bile is produced by liver hepatocytes and is stored in the gallbladder. Postprandial, the stored bile concentrated in gallbladder is drained to the duodenum to help in lipid digestion. The enterohepatic circulation of this body fluid throughout the billiary tree makes it an attractive body fluid for identification of circulatory protein biomarkers for various biliary tract cancers. Most of the studies carried out so far have characterized the bile fluid proteome only from cancer patients; thus a baseline proteome of bile from individuals without cancer of the biliary tract was not available. We carried out comprehensive proteomic profiling of bile collected from patients with calculus cholecystitis using multiple fractionation techniques coupled to high resolution Fourier transform mass spectrometry to identify more than 2500 proteins. This is the largest catalogue of any human body fluid reported in a single study till date. We are currently investigating the differential proteomics of bile obtained from calculus cholecystitis and gallbladder cancer patients using iTRAQ labeling and high resolution mass spectrometry to identify biomarkers for gallbladder cancer.


Biomarkers in Aqueous Humor

Aqueous humor (AH) is an optically clear fluid that fills the space between the lens and the cornea. This fluid is predominantly secreted by the non pigmented epithelial cells of the ciliary processes predominantly by active transport. It is a mixture of organic solutes, electrolytes, cytokines and proteins. Several reports have indicated that the protein levels in the aqueous humor vary in different ocular conditions. We are currently investigating proteomic changes seen in aqueous humor with respect to various eye-related diseases using mass spectrometry.


Biomarkers in Vitreous Humor

The vitreous humor is a transparent, gelatinous mass whose main constituent is water. It plays an important role in providing the metabolic nutrient environment for the eyes and lens, coordinating eye growth, providing support to the retina and protecting the eye from mechanical trauma. It is in close proximity to the retina and reflects many of the changes occurring in the retina. The biochemical changes occurring in the vitreous humor could provide a better understanding about the pathophysiological processes that occur in vitreoretinopathy. We investigated the proteome of normal human vitreous humor using high resolution Fourier transform mass spectrometry. Over a thousand proteins were identified, around 800 of which have not been described previously in the vitreous humor. We are investigating proteomic changes seen in vitreous humor with respect to various eye-related diseases.


Biomarkers in Ovarian Follicular Fluid

Human follicular fluid is found in the follicular antrum and surrounds the ovum in a growing ovarian follicle. It includes various biologically active proteins which affect the follicular growth and maturation and is essential for oocyte development and ovulation. This body fluid is a poorly explored fluid till date as it is a complex body fluid with proteins present in a high dynamic range. Less abundant proteins are masked by the presence of high abundant proteins that further hinder their identification. Follicular fluid analysis may serve as an alternative for ovarian biopsies to study the ovarian physiology as well as enable the investigation of the pathophysiology of several ovarian disorders. We are carrying out in depth proteomic profiling of follicular fluid from normal women and women suffering from polycystic ovary syndrome. We undertook a multifaceted approach of peptide separation coupled to high resolution Fourier transform mass spectrometry, to identify the key proteins associated with the pathogenesis of follicular cysts, some of which may be candidate biomarkers for the condition and contribute to a better understanding of the basic biology of follicle maturation.