Mass spectrometry-based proteomics has proven to be a powerful tool for studying biology, connecting genomics to cellular structure, function, signaling metabolism. In the past 20 years, the complexity of the biological questions that can be addressed using mass spectrometry has increased dramatically. Today, large scale studies allow identification of proteins in networks, as well as characterization of their post-translational modifications and expression, and give us a view of which proteins are expressed in biological networks. In the Yates lab, proteomics techniques are applied to a wide range of biological systems, and novel tools are developed to address unique challenges that arise in each application.
Cystic Fibrosis (CF) is a protein misfolding disease caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene which disturbs ion transport across a 12 transmembrane anion channel. CF affects approximately 70,000 people worldwide, making it the most common lethal genetic disorder in Caucasians. An in-frame-deletion of phenylalanine 508 (∆F508 CFTR) accounts for more than 90% of CF cases and produces a protein that is fully expressed but energetically unstable, which prevents proper folding and trafficking. However, ∆F508 CFTR channel activity can be rescued at low temperature (26-30˚C) or upon inhibition of HDAC activity (HDACi), suggesting that posttranslational processes, such as altered chaperone recruitment, are responsible for manifestation of CF. We have comprehensively characterized the wt and ∆F508 CFTR interactome and monitored CFTR interactome dynamics in order to identify interactions that are remodeled during temperature shift or upon HDACi and that potentially drive the disease phenotype. To do this we developed a novel Co-immunoprecipitation strategy and methods to analyze protein interaction specificity and dynamics which may provide a new general strategy to identify important interactors and novel drug targets.
Schizophrenia is a complex neuropsychiatric disorder which afflicts approximately 1% of the US population. It’s symptoms including hallucinations, delusions, paranoia, disorganized thoughts, social isolation and cognitive deficits, and currently there is no cure, only therapies to control a few specific symptoms. To better understand the molecular mechanisms underlying this disease, we have applied proteomic techniques to two different schizophrenia models. First, we used pairwise SILAC (stable isotope labeling by amino acids in cell culture) comparisons of hiPSC-NPC (human induced pluripotent stem cells derived from neural progenitor cells) from control and schizophrenic (SZ) patients. By using MudPIT we identified proteins that were up- or downregulated in four pairwise comparisons between gender-matched SZ (P1 and P3) and control (C1, C3 and C6) hiPSC-NPCs. Through four independent SILAC experiments, we identified perturbed proteins with corrected P-values <0.05 and >1.2-fold change in SZ hiPSC NPCs. SZ hiPSC NPCs revealed reproducible SZ-associated phenotypes, including aberrant migration and increased oxidative stress. After screening larger cohorts of SZ patients, it is hoped that these pertubations may serve as a proxy for the developmental pathways potentially contributing to SZ pathogenesis.
In a second approach, we applied quantitative phosphoproteomic analysis to a phencyclidine (PCP)-induced animal model of schizophrenia. Prepulse inhibition (PPI) is an example of sensorimotor gating, and deficits in PPI have been demonstrated in schizophrenia patients. While phencyclidine (PCP) suppression of PPI in animals has been studied to elucidate the pathological elements of schizophrenia, the molecular mechanisms underlying PCP treatment or PPI in the brain are still poorly understood. In this study, quantitative phosphoproteomic analysis using SILAM was performed on the prefrontal cortex of rats that were subjected to PPI after being systemically injected with PCP or saline. PCP treatment resulted in downregulation of phosphorylation events in the brain, and these samples were significantly enriched in proteins associated with long-term potentiation (LTP). Importantly, this data set identified functionally novel phosphorylation sites on known LTP-associated signaling molecules. In addition, mutagenesis of a significantly altered phosphorylation site on xCT (SLC7A11), the light chain of system xc-, the cystine/glutamate antiporter, suggests that PCP also regulates the activity of this protein. Finally, new insights were also derived on PPI signaling independent of PCP treatment. This was the first quantitative phosphorylation proteomic analysis providing new molecular insights into sensorimotor gating.