Lead: Indhumathy Subramaniyan
My research is mainly focused on purifying and identifying stable protein complexes using 3D-HPLC on in-vivo crosslinked whole cell lysate. In vivo cross-linking is used to stabilize transient complexes and weak interactions for analysis of Protein-Protein Interactions. Traditional methods such as IP, Western blot are useful to study a single target protein only. In-contrast, our online 3DLC - LC-MS/MS approach is capable of identifying and quantifying protein-protein interactions on a large scale.
HEK293 cells are grown in SILAC medium supplemented with heavy, light labeled amino acids. Cross-linking was performed by exposing the heavy labeled controls as well as light labeled SDA cross linked cells to UV light and followed by BN-lysis. The heavy, light lysates were mixed 1:1 and fractionated using 3DLC system. In 3DLC, we have 3 orthogonal separation columns consisting of Size-Exclusion Chromatography, Mixed-bed Ion-Exchange chromatography and Hydrophobic Interaction Chromatography. The fractionated samples are digested and analyzed using LC/MS. Using correlation analysis, known and novel protein complexes can be identified based on the co-migration of interacting partners through multiple orthogonal separations.
Our system will be used to study and characterize the Protein-Protein Interactions temporally and spatially, relative to treatment conditions or disease status.
Lead: Xiaohua Liu
Tau protein plays an important role in numerous neurological disorders, including Alzheimers disease (AD). Recent studies indicate that tau may spread between neurons in a prion-like manner, however, the molecular mechanisms involved are poorly understood. Tau is one of the most complex protein in the human brain, tau has six isoforms and forms many post-translationally modifications, such as methylation, glycosylation, ubiquitinylation, and phosphorylation. My research is focusing on characterizing and quantifying the tau proteins associate with different diseases by using mass spectrometry.
Protein biomarkers have huge potential to allow the identification of cancer subtypes, permitting the development of novel therapies and individualized prevention and treatment. However, the analysis of proteomes to identify roubust biomarkers is complex and challenging. Comprehensive identification and quantification of proteins in complex mixtures, such as tissue samples, is made possible by the development of modern separation techniques coupled to advanced mass-spectrometers. Despite improvements in the field, the identification of highly specific, low abundance protein biomarkers, which are of particular interest to scientists and clinicians, has been a major obstacle.
In breast cancer, the diversity among tumors makes the identification of proteome even more challenging. A detailed knowledge of the proteins that become activated or repressed within a tumor, post-translational modifications and comparison of their levels of expression in cancer versus normal tissue should provide insight into disease mechanisms. This would greatly help improving patient stratification and individualized disease prevention or treatment.
The project focus is to develop protein extraction methods for tissue samples, and LC-MS methods to detect and understand in detail the perturbations caused by tyrosine-kinases and other factors that in tumour versus normal breast tissue.