Brian T Chait

The Rockefeller University, New York, New York, United States

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Publications (302)2529.78 Total impact

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    ABSTRACT: The central players in most cellular events are assemblies of macromolecules. Structural and functional characterization of these assemblies requires knowledge of their subunit stoichiometry and intersubunit connectivity. One of the most direct means for acquiring such information is so-called native mass spectrometry (MS), wherein the masses of the intact assemblies and parts thereof are accurately determined. It is of particular interest to apply native MS to the study of endogenous protein assemblies-i.e., those wherein the component proteins are expressed at endogenous levels in their natural functional states rather than the overexpressed (sometimes partial) constructs commonly employed in classical structural studies, whose assembly can introduce stoichiometry artifacts and other unwanted effects. To date, the application of native MS to the elucidation of endogenous protein complexes has been limited by the difficulty in obtaining pristine cell-derived assemblies at sufficiently high concentrations for effective analysis. To address this challenge, we present here a robust workflow that couples rapid and efficient affinity isolation of endogenous protein complexes with a sensitive native MS readout. The resulting workflow has the potential to provide a wealth of data on the stoichiometry and intersubunit connectivity of endogenous protein assemblies-information that is key to successful integrative structural elucidation of biological systems.
    No preview · Article · Feb 2016 · Analytical Chemistry
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    ABSTRACT: Antibodies made in large animals are integral to many biomedical research endeavors. Domesticated herd animals like goats, sheep, donkeys, horses and camelids all offer distinct advantages in antibody production. However, their cost of use is often prohibitive, especially where poor antigen response is commonplace; choosing a non-responsive animal can set a research program back or even prevent experiments from moving forward entirely. Over the course of production of antibodies from llamas, we found that some animals consistently produced a higher humoral antibody response than others, even to highly divergent antigens, as well as to their standard vaccines. Based on our initial data, we propose that these “high level responders” could be pre-selected by checking antibody titers against common vaccines given to domestic farm animals. Thus, time and money can be saved by reducing the chances of getting poor responding animals and minimizing the use of superfluous animals.
    No preview · Article · Jan 2016 · Journal of immunological methods
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    ABSTRACT: At the eukaryotic DNA replication fork, it is widely believed that the Cdc45-Mcm2-7-GINS (CMG) helicase is positioned in front to unwind DNA and that DNA polymerases trail behind the helicase. Here we used single-particle EM to directly image a Saccharomyces cerevisiae replisome. Contrary to expectations, the leading strand Pol ɛ is positioned ahead of CMG helicase, whereas Ctf4 and the lagging-strand polymerase (Pol) α-primase are behind the helicase. This unexpected architecture indicates that the leading-strand DNA travels a long distance before reaching Pol ɛ, first threading through the Mcm2-7 ring and then making a U-turn at the bottom and reaching Pol ɛ at the top of CMG. Our work reveals an unexpected configuration of the eukaryotic replisome, suggests possible reasons for this architecture and provides a basis for further structural and biochemical replisome studies.
    Full-text · Article · Nov 2015 · Nature Structural & Molecular Biology
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    ABSTRACT: Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that are activated by growth factor and G-protein coupled receptors, and propagate intracellular signals for growth, survival, proliferation, and metabolism. p85α, a modular protein consisting of 5 domains, binds and inhibits the enzymatic activity of Class IA PI3Ks. Here, we describe the structural states of the p85α dimer, based on data from in vivo and in vitro solution characterization. Our in vitro assembly and structural analyses have been enabled by the creation of cysteine-free p85α that is functionally equivalent to native p85α. Analytical ultracentrifugation (AUC) studies showed that p85α undergoes rapidly reversible monomer-dimer assembly that is highly exothermic in nature. In addition to the documented SH3-PR1 dimerization interaction, we identified a second intermolecular interaction mediated by cSH2 domains at the C-terminal end of the polypeptide with unique electrostatic character. We have demonstrated in vivo concentration-dependent dimerization of p85α using fluorescence fluctuation spectroscopy (FFS). Finally, we have defined solution conditions under which the protein is predominantly monomeric or dimeric, providing the basis for small angle X-ray scattering (SAXS) and chemical cross-linking structural analysis of the discrete dimer. These experimental data are used for the integrative structure determination of the p85α dimer. Our study provides new insight into the structure and assembly of the p85α homodimer and suggests that this protein is a highly dynamic molecule whose conformational flexibility allows it to transiently associate with multiple binding proteins.
    Full-text · Article · Oct 2015 · Journal of Biological Chemistry
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    ABSTRACT: It remains particularly problematic to define the structures of native macromolecular assemblies, which are often of low abundance. Here we present a strategy for isolating complexes at endogenous levels from GFP-tagged transgenic cell lines. Using cross-linking mass spectrometry, we extracted distance restraints that allowed us to model the complexes' molecular architectures.
    Full-text · Article · Oct 2015 · Nature Methods
  • Deanna H. Morris · Calvin K. Yip · Yi Shi · Brian T. Chait · Qing Jun Wang
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    ABSTRACT: Autophagy is an important lysosomal degradation pathway that aids in the maintenance of cellular homeostasis by breaking down and recycling intracellular contents. Dysregulation of autophagy is linked to a growing number of human diseases. The Beclin 1-Vps34 protein-protein interaction network is critical for autophagy regulation and is therefore essential to cellular integrity. Manipulation of autophagy, in particular via modulation of the action of the Beclin 1-Vps34 complexes, is considered a promising route to combat autophagy-related diseases. Here we summarize recent findings on the core components and structural architecture of the Beclin 1-Vps34 complexes, and how these findings provide valuable insights into the molecular mechanisms that underlie the multiple functions of these complexes and for devising therapeutic strategies. © 2015, Higher Education Press and Springer-Verlag Berlin Heidelberg.
    No preview · Article · Oct 2015
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    ABSTRACT: DNA double-strand break repair involves phosphorylation of histone variant H2AX ('γH2AX'), which accumulates in foci at sites of DNA damage. In current models, the recruitment of multiple DNA repair proteins to γH2AX foci depends mainly on recognition of this 'mark' by a single protein, MDC1. However, DNA repair proteins accumulate at γH2AX sites without MDC1, suggesting that other 'readers' of this mark exist. Here, we use a quantitative chemical proteomics approach to profile direct, phospho-selective γH2AX binders in native proteomes. We identify γH2AX binders, including the DNA repair mediator 53BP1, which we show recognizes γH2AX through its BRCT domains. Furthermore, we investigate the targeting of wild-type 53BP1, or a mutant form deficient in γH2AX binding, to chromosomal breaks resulting from endogenous and exogenous DNA damage. Our results show how direct recognition of γH2AX modulates protein localization at DNA damage sites, and suggest how specific chromatin mark-reader interactions contribute to essential mechanisms ensuring genome stability.
    No preview · Article · Sep 2015 · Nature Chemical Biology
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    Full-text · Dataset · Aug 2015
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    ABSTRACT: β-lactoglobulin (BLG) present in cow's milk is one of the most aggressive allergens for the newborns. In exclusively breastfed infants, cow’s milk protein allergy is triggered by mother's diet. Since no prophylactic treatment is available, binding of BLG with a ligand able to reduce the allergenicity is currently in the focus of research. In the present work, BLG extracted from human milk following a deliberate intake of cow's milk was subjected to a noncovalent interaction with maltohexaose (Glc6). The reaction products were identified and structurally analyzed. An advanced mass spectrometric (MS) platform based on fully automated chip nanoelectrospray (nanoESI) was developed and optimized for this purpose. Employment of fully automated chip nanoESI, which allowed working in high throughput mode, was crucial for the assessment of the interaction products almost in real time, without jeopardizing the reaction conditions. The experiments were carried out in aq. ammonium acetate/formic acid (pH 6,0) buffer and revealed the formation of BLG-maltohexaose (Glc6) complex, which was characterized in details by top-down fragmentation using collison-induced dissociation (CID). According to the obtained data, the BLG-Glc6 noncovalent binding is a very fast process. Within only one minute of reaction, the complex was already formed. The mass spectra of the reaction products collected each 5 minutes over a half an hour revealed a significant increase in the abundance of the corresponding molecular ion. Our findings have major clinical impact, indicating that maltooligosaccharides represent a class of ligands that bind BLG and therefore might reduce its allergenicity.
    No preview · Conference Paper · Jun 2015
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    ABSTRACT: Cow's milk protein allergy in exclusively breastfed infants, the main cause of food intolerance during the first 6 months of life, is triggered by the mother's diet. β-Lactoglobulin (BLG) present in cow's milk is one of the most potent allergens for newborns. Since no prophylactic treatment is available, finding ligands capable of binding BLG and reducing its allergenicity is currently the focus of research. In this work, an innovative methodology encompassing microfluidics based on fully automated chip-nanoelectrospray ionization (nanoESI), coupled with high-resolution mass spectrometry (MS) on a quadrupole time-of-flight (QTOF MS) instrument was developed. This platform was employed for the assessment of the noncovalent interactions between maltohexaose (Glc6) and β-lactoglobulin extracted from human milk upon deliberate intake of cow's milk. The experiments were carried out in (+) ESI mode, using ammonium acetate (pH 6.0) as the buffer and also in pure water. In both cases, the MS analysis revealed the formation of BLG-Glc6 complex, which was characterized by top-down fragmentation in tandem MS (MS/MS) using collision-induced dissociation (CID). Our findings have a significant biomedical impact, indicating that Glc6 binds BLG under conditions mimicking the in vivo environment and therefore might represent a ligand, able to reduce its allergenicity.
    Full-text · Article · Jun 2015 · Amino Acids
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    ABSTRACT: Breast cancer is nowadays one of the most severe forms of cancer, being a major cause of women mortality worldwide. The most common diagnostic method is based on mammography, which like all imaging methods has several limitations; the breast tumor is sometimes less visible and can be detected by mammography only when the tumor has reached life-threatening dimensions. Modern proteomics based on mass spectrometry (MS) and allied techniques such as gel electrophoresis (GE) may represent a reliable methodology for early detection of breast cancer through specific biomarkers that can be detected in the milk secreted by the affected breast tissue. We have analyzed milk aliquots from healthy women and from women diagnosed with breast cancer. The milk proteins were run by SDS-PAGE. A comparative assay healthy vs. cancerous breast has been carried out, using as samples milk aliquots from age and race-matched patients and controls. Our preliminary results indicate that proteomics based methodologies represent a viable tool in early detection and assessment of breast cancer condition. Our preliminary results indicate that proteomics based methodologies represent a viable tool in early detection and assessment of breast cancer condition. Further studies will be certainly undertaken for development of this method and its introduction as a routine diagnostic procedure in this type of disease. Novel protein biomarkers of breast cancer were identified in human milk using a complex approach based on PAGE and nanoLC-MS/MS.
    No preview · Conference Paper · Jun 2015
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    ABSTRACT: We must reliably map the interactomes of cellular macromolecular complexes in order to fully explore and understand biological systems. However, there are no methods to accurately predict how to capture a given macromolecular complex with its physiological binding partners. Here, we present a screen that comprehensively explores the parameters affecting the stability of interactions in affinity-captured complexes, enabling the discovery of physiological binding partners and the elucidation of their functional interactions in unparalleled detail. We have implemented this screen on several macromolecular complexes from a variety of organisms, revealing novel profiles even for well-studied proteins. Our approach is robust, economical and automatable, providing an inroad to the rigorous, systematic dissection of cellular interactomes.
    No preview · Article · May 2015 · Nature Methods
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    ABSTRACT: Surface membrane organization and composition is key to cellular function, and membrane proteins serve many essential roles in endocytosis, secretion and cell recognition. The surface of parasitic organisms, however, is a double-edged sword; this is the primary interface between parasites and their hosts, and those crucial cellular processes must be carried out while avoiding elimination by the host immune defenses. For extracellular African trypanosomes, the surface is partitioned such that all endo- and exocytosis is directed through a specific membrane region, the flagellar pocket, in which it is thought the majority of invariant surface proteins reside. However, very few of these proteins have been identified, severely limiting functional studies, and hampering the development of potential treatments. Here we used an integrated biochemical, proteomic and bioinformatic strategy to identify surface components of the human parasite Trypanosoma brucei. This surface proteome contains previously known flagellar pocket proteins as well as multiple novel components, and is significantly enriched in proteins that are essential for parasite survival. Molecules with receptor-like properties are almost exclusively parasite-specific, whereas transporter-like proteins are conserved in model organisms. Validation shows that the majority of surface proteome constituents are bona fide surface-associated proteins, and as expected, the majority present at the flagellar pocket. Moreover, the largest systematic analysis of trypanosome surface molecules to date provides evidence that the cell surface is compartmentalized into three distinct domains with free diffusion of molecules in each, but selective, asymmetric traffic between. This work provides a paradigm for the compartmentalization of a cell surface and a resource for its analysis. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    No preview · Article · Apr 2015 · Molecular & Cellular Proteomics
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    ABSTRACT: Breast cancer is nowadays one of the most severe forms of cancer, being a major cause of women mortality worldwide. The most common diagnostic method is based on mammography, which like all imaging methods has several limitations; the breast tumor is sometimes less visible and can be detected by mammography only when the tumor has reached life-threatening dimensions. Modern proteomics based on mass spectrometry (MS) and allied techniques such as gel electrophoresis (GE) may represent a reliable methodology for early detection of breast cancer through specific biomarkers that can be detected in the milk secreted by the affected breast tissue.
    No preview · Conference Paper · Mar 2015
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    ABSTRACT: Dissecting and studying cellular systems requires the ability to specifically isolate distinct proteins along with the co-assembled constituents of their associated complexes. Affinity capture techniques leverage high affinity, high specificity reagents to target and capture proteins of interest along with specifically associated proteins from cell extracts. Affinity capture coupled to mass spectrometry (MS)-based proteomic analyses has enabled the isolation and characterization of a wide range of endogenous protein complexes. Here, we outline effective procedures for the affinity capture of protein complexes, highlighting best practices and common pitfalls.
    Full-text · Article · Mar 2015 · BioTechniques
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    ABSTRACT: We demonstrate that the efficiency of ion transmission from atmosphere to vacuum through stainless steel electrodes that contain slowly divergent conical duct (ConDuct) channels can be close to 100%. Here, we explore the properties of 2.5-cm-long electrodes with angles of divergence of 0°, 1°, 2°, 3°, 5°, 8°, 13°, and 21°, respectively. The ion transmission efficiency was observed to jump from 10-20% for the 0° (straight) channels to 90-95% for channels with an angle of divergence as small as 1°. Furthermore, the 2-3° ConDuct electrodes produced extraordinarily low divergence ion beams that propagated in a laser-like fashion over long distances in vacuum. To take advantage of these newly discovered properties, we constructed a novel atmosphere-to-vacuum ion interface utilizing a 2° ConDuct as an inlet electrode and compared its ion transmission efficiency with that of the interface used in the commercial (Thermo Fisher Scientific, San Jose, CA, USA) Velos Orbitrap and Q Exactive mass spectrometers. We observed that the ConDuct interface transmitted up to 17 times more ions than the commercial reference interface and also yielded improved signal-to-noise mass spectra of peptides. We infer from these results that the performance of many current atmosphere-to-vacuum interfaces utilizing metal capillaries can be substantially improved by replacing them with 1° or 2° metal ConDuct electrodes, which should preserve the convenience of supplying ion desolvation energy by heating the electrode while greatly increasing the efficiency of ion transmission into the mass spectrometer.
    No preview · Article · Feb 2015 · Journal of the American Society for Mass Spectrometry
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    ABSTRACT: Metabotropic glutamate receptor 5 (mGluR5) regulates excitatory postsynaptic signaling in the central nervous system (CNS) and is implicated in various CNS disorders. Protein kinase A (PKA) signaling is known to play a critical role in neuropsychiatric disorders such as Parkinson's disease, schizophrenia and addiction. Dopamine signaling is known to modulate the properties of mGluR5 in a cAMP- and PKA-dependent manner, suggesting that mGluR5 may be a direct target for PKA. Our study identifies mGluR5 at Ser870 as a direct substrate for PKA phosphorylation and demonstrates that this phosphorylation plays a critical role in the PKA-mediated modulation of mGluR5 functions such as extracellular signal-regulated kinase (ERK) phosphorylation and intracellular Ca(2+) oscillations. The identification of the molecular mechanism by which PKA signaling modulates mGluR5-mediated cellular responses contributes to the understanding of the interaction between dopaminergic and glutamatergic neuronal signaling. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    No preview · Article · Feb 2015 · Journal of Neurochemistry
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    ABSTRACT: We have discovered that an electrode containing a conical channel with a small angular divergence can transmit into the vacuum almost 100% of an electrospray ion current produced at atmospheric pressure. Our first implementation of such a conical duct, which we term "ConDuct," uses a conductive plastic pipette tip containing an approximately 1.6° divergent channel at its entrance. We observed that the beam formed by the ConDuct electrode has a very low divergence (less than 1°) and persists for long distances in vacuum. Intrigued by these properties, we incorporated this electrode into a novel atmosphere-to-vacuum ion transmission interface, and devised a technique for evaluating its performance relative to the commercial reference interfaces that contain heated metal capillaries. We determined that our new interface transmits at least 400 times more ions than the commercial Thermo LCQ DECA XP atmosphere-to-vacuum interface and 2 to 3 times more than the commercial interface in the Thermo Velos Orbitrap and the Q Exactive mass spectrometers. We conclude that it might be possible to optimize the properties of the transmitted ions further by manufacturing ConDuct inlet electrodes from metal rather than conductive plastic and by determining the optimum angle of channel divergence and channel length.
    No preview · Article · Jan 2015 · Journal of the American Society for Mass Spectrometry
  • Mirco Sorci · Ryo Hayama · Brian T. Chait · Michael Rout · Georges Belfort
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    ABSTRACT: Membrane fouling, the process in which proteins adhere to the external and internal surface of synthetic membranes, is considered a major limitation in membrane filtration of biofluids. This causes permeation flux and changes in selectivity, with the overall effect of reducing membrane performance and increasing process cost. In the past 30 years researchers have focused on how to reduce membrane fouling. The Whitesides group has published a set of criteria for obtaining protein-resistant chemistries, viz. the surface of such membranes or materials should (i) be hydrophilic, (ii) include hydrogen bond acceptors, (iii) do not include hydrogen bond donors, and (iv) be net electrically neutral. An alternative approach to producing synthetic surfaces based on these criteria is to observe Nature’s method of producing protein-resistant surfaces. In particular, we focused on the Nuclear Pore Complex (NPC), the sole mediator of exchange between the nucleus and the cytoplasm in all eukaryotic cells. Protein transport across the NPC is fast, energy-dependent (to give directionality), often receptor-mediated and most important it occurs in the absence of unwanted fouling. For this reason we focused on “NPC-proteins” and covalently attached them on a sensor surface to test their antifouling properties. A series of different proteins, including BSA, were used to test the efficacy of this new biomimetic surface. For comparison (control), we also tested surfaces modified with other inert proteins and PEG, the gold standard with respect to protein-resistance. A quartz crystal microbalance with dissipation was used in all these experiments.
    No preview · Conference Paper · Nov 2014
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    ABSTRACT: The evolutionarily conserved Mediator complex is a critical coactivator for RNA polymerase II (Pol II)-mediated transcription. Here we report the reconstitution of a functional 15-subunit human core Mediator complex and its characterization by functional assays and chemical cross-linking coupled to MS (CX-MS). Whereas the reconstituted head and middle modules can stably associate, basal and coactivator functions are acquired only after incorporation of MED14 into the bimodular complex. This results from a dramatically enhanced ability of MED14-containing complexes to associate with Pol II. Altogether, our analyses identify MED14 as both an architectural and a functional backbone of the Mediator complex. We further establish a conditional requirement for metazoan-specific MED26 that becomes evident in the presence of heterologous nuclear factors. This general approach paves the way for systematic dissection of the multiple layers of functionality associated with the Mediator complex.
    Full-text · Article · Nov 2014 · Nature Structural & Molecular Biology

Publication Stats

30k Citations
2,529.78 Total Impact Points

Institutions

  • 1985-2015
    • The Rockefeller University
      • • Laboratory of Mass Spectrometry and Gaseous Ion Chemistry
      • • Laboratory of Cellular and Structural Biology
      • • Laboratory of Molecular Neurobiology and Biophysics
      New York, New York, United States
  • 2010
    • Princeton University
      • Department of Molecular Biology
      Princeton, NJ, United States
  • 1987-2010
    • CUNY Graduate Center
      New York, New York, United States
  • 2001
    • Baylor College of Medicine
      Houston, Texas, United States
  • 1998
    • Brandeis University
      Волтам, Massachusetts, United States