Proteomic methods continue to evolve at a rapid pace. Advances in instrumentation, sample preparation, and mass spectrometric methodology are constant. At the heart of our analysis pipeline is a Thermo Scientific Orbitrap Fusion Tribrid mass spectrometer. This high mass resolution instrument, with three separate mass filters, is incredibly versatile, sensitive, and fast. It allows us to routinely quantify >8000 proteins from up to 10 different samples in a single experiment. Finding ways to apply this technology to novel biological problems and increase the depth and accuracy of our analysis is a major focus of the lab. We are currently developing an untargeted platform for monitoring changes in protein-protein interactions.
Understanding the roles of protein phosphorylation
One of the areas in which mass spectrometry has made the greatest impact is in the identification of post-translationally modified proteins and the localization of these modifications to specific amino acid residues. Proteins can be modified by the addition of phosphoryl, acetyl, methyl, and many other chemical groups as well as by isopeptide linkages to other proteins such as ubiquitin and SUMO. These modifications can have profound impacts on cellular proteins and cellular physiology; they are used to activate and deactivate enzymes, control sub-cellular localization, mark proteins for degradation, direct protein trafficking, and modulate protein-protein interactions. Through these and other functions, they regulate nearly every aspect of cellular growth and proliferation, as well as cellular responses to changing environmental conditions. In recent years the application of mass spectrometric methods has generated an explosion of data identifying many thousands of protein phosphorylation, acetylation, and ubiquitylation sites. However, the details and physiological relevance of the vast majority of these sites is completely unknown. Uncovering these relationships is one of the main goals of our research. We use multiplexed quantitative phosphoproteomics in vivo and in vitro to identify protein kinase substrates and downstream signaling events in different cellular contexts. Parallel analyses of protein interactions and localization using proteomic and classical biochemical and cell biological methods allow us to identify sites that affect cellular physiology.
Multi-proteomic approaches to understand signaling in the epithelial to mesenchymal transition
EMT is a program of epithelial cell dedifferentiation in which cells growing as components of a highly polarized monolayer lose cell-cell contact and escape as non-polarized cells capable of moving to other tissues and differentiating into multiple cell types. EMT was first observed in metazoan embryogenesis, but is now known to play complex roles in tumor progression. EMT contributes to the acquisition of invasive and metastatic properties, contributes to drug resistance, and promotes the generation of cancer stem cells. Despite the biological importance of EMT in development and disease and the potential therapeutic and diagnostic value of properly understanding EMT, a modern global and quantitative analysis of the molecular mechanisms responsible for these changes, and of the specific roles of the numerous signaling pathways proposed to be involved is still missing. Using multiplexed TMT analysis and biochemical cell fractionation we are generating a proteome-wide combined spatiotemporal and phosphoproteomic map of cells as they pass through EMT.
The consequences of aneuploidy
Aneuploidy, an abnormal number of chromosomes, causes severe developmental defects and is a near universal feature of tumor cells. Despite its profound effects, the cellular processes affected by aneuploidy are not well characterized. In collaboration with Eduardo Torres at the University of Massachusetts Medical School, we are using a yeast model to investigate the molecular consequences of aneuploidy by examining changes in the proteome in a collection of aneuploid strains. These studies have already produced a number of intriguing results. Though levels of most proteins coded on extra chromosomes increase proportionally with gene copy number, approximately 20% of duplicated genes are dosage compensated. These dosage compensated genes are heavily enriched for elements of multi-protein complexes. Surprisingly, these compensated proteins are being transcribed and translated at gene-copy proportional levels - thus compensation is mediated posttranslationally. We postulate that excess protein subunits that are not incorporated into their protein complexes are rapidly degraded.