Faculty Bibliography

Labeling peptides with isobaric tags is a popular strategy in quantitative bottom-up proteomics. In this study, we labeled six breast tumor cell lysates (1.34 mg proteins per channel) using 10-plex tandem mass tag reagents and analyzed the samples on a Q Exactive HF Quadrupole-Orbitrap mass spectrometer. We identified a total of 8,706 proteins and 28,186 phosphopeptides, including 7,394 proteins and 23,739 phosphosites common to all channels. The majority of technical replicates correlated with a R2 >/= 0.98, indicating minimum variability was introduced after labeling. Unsupervised hierarchical clustering of phosphopeptide data sets successfully classified the breast tumor samples into Her2 (epidermal growth factor receptor 2) positive and Her2 negative groups, whereas mRNA abundance did not. The tyrosine phosphorylation levels of receptor tyrosine kinases, phosphoinositide-3-kinase, protein kinase C delta, and Src homology 2, among others, were significantly higher in the Her2 positive than the Her2 negative group. Despite ratio compression in MS2-based experiments, we demonstrated the ratios calculated using an MS2 method are highly correlated (R2 > 0.65) with ratios obtained using MS3-based quantitation (using a Thermo Orbitrap Fusion mass spectrometer) with reduced ratio suppression. Given the deep coverage of global and phosphoproteomes, our data show that MS2-based quantitation using TMT can be successfully used for large-scale multiplexed quantitative proteomics.

Receptor tyrosine kinase (RTK) signaling is tightly regulated by protein allostery within the intracellular tyrosine kinase domains. Yet the molecular determinants of allosteric connectivity in tyrosine kinase domain are incompletely understood. By means of structural (X-ray and NMR) and functional characterization of pathogenic gain-of-function mutations affecting the FGF receptor (FGFR) tyrosine kinase domain, we elucidated a long-distance allosteric network composed of four interconnected sites termed the 'molecular brake', 'DFG latch', 'A-loop plug', and 'alphaC tether'. The first three sites repress the kinase from adopting an active conformation, whereas the alphaC tether promotes the active conformation. The skewed design of this four-site allosteric network imposes tight autoinhibition and accounts for the incomplete mimicry of the activated conformation by pathogenic mutations targeting a single site. Based on the structural similarity shared among RTKs, we propose that this allosteric model for FGFR kinases is applicable to other RTKs.

Detection of differentially abundant proteins in label-free quantitative shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS) experiments requires a series of computational steps that identify and quantify LC-MS features. It also requires statistical analyses that distinguish systematic changes in abundance between conditions from artifacts of biological and technical variation. The 2015 study of the Proteome Informatics Research Group (iPRG) of the Association of Biomolecular Resource Facilities (ABRF) aimed to evaluate the effects of the statistical analysis on the accuracy of the results. The study used LC-tandem mass spectra acquired from a controlled mixture, and made the data available to anonymous volunteer participants. The participants used methods of their choice to detect differentially abundant proteins, estimate the associated fold changes, and characterize the uncertainty of the results. The study found that multiple strategies (including the use of spectral counts versus peak intensities, and various software tools) could lead to accurate results, and that the performance was primarily determined by the analysts' expertise. This manuscript summarizes the outcome of the study, and provides representative examples of good computational and statistical practice. The data set generated as part of this study is publicly available.

Reduced amino acid availability attenuates mRNA translation in cells and helps to extend lifespan in model organisms. The amino acid deprivation-activated kinase GCN2 mediates this response in part by phosphorylating eIF2alpha. In addition, the cap-dependent translational inhibitor 4E-BP is transcriptionally induced to extend lifespan in Drosophila melanogaster, but through an unclear mechanism. Here, we show that GCN2 and its downstream transcription factor, ATF4, mediate 4E-BP induction, and GCN2 is required for lifespan extension in response to dietary restriction of amino acids. The 4E-BP intron contains ATF4-binding sites that not only respond to stress but also show inherent ATF4 activity during normal development. Analysis of the newly synthesized proteome through metabolic labeling combined with click chemistry shows that certain stress-responsive proteins are resistant to inhibition by 4E-BP, and gcn2 mutant flies have reduced levels of stress-responsive protein synthesis. These results indicate that GCN2 and ATF4 are important regulators of 4E-BP transcription during normal development and aging.

Aims Several lines of investigation support the notion that synaptic pathology, one of the strongest correlates to cognitive impairment, is related to progressive accumulation of neurotoxic amyloid beta (Abeta) oligomers. Since the process of oligomerization/fibrillization is concentration-dependent, it is highly reliant on the homeostatic mechanisms that regulate the steady state levels of Abeta influencing the delicate balance between rate of synthesis, dynamics of aggregation and clearance kinetics. Emerging new data suggest that reduced Abeta clearance, particularly in the aging brain, plays a critical role in the process of amyloid formation and AD pathogenesis. Method We have used a combination of stereotaxic injection into the hippocampal region of C57BL/6 wild-type mice with biochemical and mass spectrometric analyses of CSF to evaluate the brain clearance and catabolism of well-defined monomeric and low molecular mass Abeta oligomeric assemblies. Results Abeta physiologic removal from the brain is extremely fast, involves local proteolytic degradation with generation of heterogeneous C-terminally cleaved proteolytic products, and is negatively influenced by oligomerization. Immunofluorescence confocal microscopy studies provide insight into the cellular pathways involved in the brain removal and cellular uptake of Abeta. Clearance from brain interstitial fluid follows local and systemic paths; in addition to the BBB, local enzymatic degradation and transport through the choroid plexus into the CSF play significant roles. Conclusion Our studies highlight the diverse factors influencing brain clearance and the participation of various routes of elimination opening up new research opportunities for the understanding of altered mechanisms triggering AD pathology and for the potential design of combined therapeutic strategies

Cardiolipin is a specific mitochondrial phospholipid that has a high affinity for proteins and that stabilizes the assembly of supercomplexes involved in oxidative phosphorylation. We found that sequestration of cardiolipin in protein complexes is critical to protect it from degradation. The turnover of cardiolipin is slower by almost an order of magnitude than the turnover of other phospholipids. However, in subjects with Barth syndrome, cardiolipin is rapidly degraded via the intermediate monolyso-cardiolipin. Treatments that induce supercomplex assembly decrease the turnover of cardiolipin and the concentration of monolyso-cardiolipin, whereas dissociation of supercomplexes has the opposite effect. Our data suggest that cardiolipin is uniquely protected from normal lipid turnover by its association with proteins, but this association is compromised in subjects with Barth syndrome, leading cardiolipin to become unstable, which in turn causes the accumulation of monolyso-cardiolipin.

The misfolding and accumulation of the protein fragment beta-amyloid (Abeta) is an early and essential event in the pathogenesis of Alzheimer's disease (AD). Despite close biological similarities among primates, humans appear to be uniquely susceptible to the profound neurodegeneration and dementia that characterize AD, even though nonhuman primates deposit copious Abeta in senile plaques and cerebral amyloid-beta angiopathy as they grow old. Because the amino acid sequence of Abeta is identical in all primates studied to date, we asked whether differences in the properties of aggregated Abeta might underlie the vulnerability of humans and the resistance of other primates to AD. In a comparison of aged squirrel monkeys (Saimiri sciureus) and humans with AD, immunochemical and mass spectrometric analyses indicate that the populations of Abeta fragments are largely similar in the 2 species. In addition, Abeta-rich brain extracts from the brains of aged squirrel monkeys and AD patients similarly seed the deposition of Abeta in a transgenic mouse model. However, the epitope exposure of aggregated Abeta differs in sodium dodecyl sulfate-stable oligomeric Abeta from the 2 species. In addition, the high-affinity binding of 3H Pittsburgh Compound B to Abeta is significantly diminished in tissue extracts from squirrel monkeys compared with AD patients. These findings support the hypothesis that differences in the pathobiology of aggregated Abeta among primates are linked to post-translational attributes of the misfolded protein, such as molecular conformation and/or the involvement of species-specific cofactors.