Thomas A. Neubert, Ph.D.

RNA binding proteins accumulate at the postsynaptic density with synaptic activity
Zhang, Guoan; Neubert, Thomas A; Jordan, Bryen A
2012 Jan 11;32(2):599-609, Journal of neuroscience
Neuronal activity elicits changes in synaptic composition that play an important role in experience-dependent plasticity (Choquet and Triller, 2003; Lisman and Raghavachari, 2006; Bourne and Harris, 2008; Holtmaat and Svoboda, 2009). We used a modified version of stable isotope labeling by amino acids in cell culture to identify activity-dependent modifications in the composition of postsynaptic densities (PSDs) isolated from rat primary neuronal cultures. We found that synaptic activity altered approximately 2% of the PSD proteome, which included an increase in diverse RNA binding proteins (RNABPs). Indeed, 12 of the 37 identified proteins whose levels changed with synaptic activity were RNABPs and included the heterogeneous nuclear ribonucleoproteins (hnRNPs) G, A2/B1, M, and D. Knockdown of hnRNPs M and G using shRNAs resulted in altered numbers of dendritic spines, suggesting a crucial role for these proteins in spine density. Synaptic activity also resulted in a concomitant increase in dendritic and synaptic poly(A) mRNA. However, this increase was not affected by knockdown of hnRNPs M or G. Our results suggest that hnRNP proteins regulate dendritic spine density and may play a role in synaptodendritic mRNA metabolism
— id: 149813, year: 2012, vol: 32, page: 599, stat: Journal Article,

Identifying transient protein-protein interactions in EphB2 signaling by blue native PAGE and mass spectrometry
Darie, Costel C; Deinhardt, Katrin; Zhang, Guoan; Cardasis, Helene S; Chao, Moses V; Neubert, Thomas A
2011 Dec;11(23):4514-4528, Proteomics
Receptor tyrosine kinases (RTKs) are proteins that upon ligand stimulation undergo dimerization and autophosphorylation. Eph receptors (EphRs) are RTKs that are found in different cell types, from both tissues that are developing and from mature tissues, and play important roles in the development of the central nervous system and peripheral nervous system. EphRs also play roles in synapse formation, neural crest formation, angiogenesis and in remodeling the vascular system. Interaction of EphRs with their ephrin ligands lead to activation of signal transduction pathways and formation of many transient protein-protein interactions that ultimately leads to cytoskeletal remodeling. However, the sequence of events at the molecular level is not well understood. We used blue native PAGE and MS to analyze the transient protein-protein interactions that resulted from the stimulation of EphB2 receptors by their ephrinB1-Fc ligands. We analyzed the phosphotyrosine-containing protein complexes immunoprecipitated from the cell lysates of both unstimulated (-) and ephrinB1-Fc-stimulated (+) NG108 cells. Our experiments allowed us to identify many signaling proteins, either known to be part of EphB2 signaling or new for this pathway, which are involved in transient protein-protein interactions upon ephrinB1-Fc stimulation. These data led us to investigate the roles of proteins such as FAK, WAVEs and Nischarin in EphB2 signaling
— id: 145796, year: 2011, vol: 11, page: 4514, stat: Journal Article,

Neuronal growth cone retraction relies on proneurotrophin receptor signaling through rac
Deinhardt, Katrin; Kim, Taeho; Spellman, Daniel S; Mains, Richard E; Eipper, Betty A; Neubert, Thomas A; Chao, Moses V; Hempstead, Barbara L
2011 ;4(202):ra82-ra82, Science signaling
Growth of axons and dendrites is a dynamic process that involves guidance molecules, adhesion proteins, and neurotrophic factors. Although neurite extension is stimulated by the neurotrophin nerve growth factor (NGF), we found that the precursor of NGF, proNGF, induced acute collapse of growth cones of cultured hippocampal neurons. This retraction was initiated by an interaction between the p75 neurotrophin receptor (p75(NTR)) and the sortilin family member SorCS2 (sortilin-related VPS10 domain-containing receptor 2). Binding of proNGF to the p75(NTR)-SorCS2 complex induced growth cone retraction by initiating the dissociation of the guanine nucleotide exchange factor Trio from the p75(NTR)-SorCS2 complex, resulting in decreased Rac activity and, consequently, growth cone collapse. The actin-bundling protein fascin was also inactivated, contributing to the destabilization and collapse of actin filaments. These results identify a bifunctional signaling mechanism by which proNGF regulates actin dynamics to acutely modulate neuronal morphology
— id: 146263, year: 2011, vol: 4, page: ra82, stat: Journal Article,

Cardiac ATP-sensitive K+ channel associates with the glycolytic enzyme complex
Hong, Miyoun; Kefaloyianni, Eirini; Bao, Li; Malester, Brian; Delaroche, Diane; Neubert, Thomas A; Coetzee, William A
2011 Jul;25(7):2456-2467, FASEB journal
Being gated by high-energy nucleotides, cardiac ATP-sensitive potassium (K(ATP)) channels are exquisitely sensitive to changes in cellular energy metabolism. An emerging view is that proteins associated with the K(ATP) channel provide an additional layer of regulation. Using putative sulfonylurea receptor (SUR) coiled-coil domains as baits in a 2-hybrid screen against a rat cardiac cDNA library, we identified glycolytic enzymes (GAPDH and aldolase A) as putative interacting proteins. Interaction between aldolase and SUR was confirmed using GST pulldown assays and coimmunoprecipitation assays. Mass spectrometry of proteins from K(ATP) channel immunoprecipitates of rat cardiac membranes identified glycolysis as the most enriched biological process. Coimmunoprecipitation assays confirmed interaction for several glycolytic enzymes throughout the glycolytic pathway. Immunocytochemistry colocalized many of these enzymes with K(ATP) channel subunits in rat cardiac myocytes. The catalytic activities of aldolase and pyruvate kinase functionally modulate K(ATP) channels in patch-clamp experiments, whereas d-glucose was without effect. Overall, our data demonstrate close physical association and functional interaction of the glycolytic process (particularly the distal ATP-generating steps) with cardiac K(ATP) channels.-Hong, M., Kefaloyianni, E., Bao, L., Malester, B., Delaroche, D., Neubert, T. A., Coetzee, W. A. Cardiac ATP-sensitive K(+) channel associates with the glycolytic enzyme complex
— id: 134908, year: 2011, vol: 25, page: 2456, stat: Journal Article,

Catabolism of Alzheimer's amyloid-b: Implications for brain clearance and plaque deposition
McIntee F.L.; Giannoni P.; Blais S.; Neubert T.; Mathews P.; Rostagno A.; Ghiso J.
2011 ;8:?-?, Neuro-degenerative diseases
Alzheimer's disease (AD) is the leading cause of dementia and the most common form of amyloidosis in humans. Extensive extracellular deposition of amyloid-beta (Abeta), a 40-42 amino acid degradation product of APP, is considered a hallmark feature of AD. Our attention is focused on the highly heterogeneous biochemical nature of the brain Abeta species, delving beyond Abeta40 and Abeta42, likely reflecting a complex balance between amyloidogenic and clearance pathways. We have fractionated water-soluble, detergent-soluble and formic acid soluble Abeta species from brains of transgenic mouse models of amyloid depostion and AD cases. Subsequently, we applied a combination of biochemical techniques including immunoprecipitation followed by identification of Abeta species with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Our biochemical data on the Abeta species present in sporadic AD cases and in transgenic mouse models highlight the presence of similar N-and C-terminally truncated fragments-likely reflecting the ability of multiple proteases to degrade Abeta in situ-and several post-translational modifications with still unclear roles in the amyloidogenesis mechanism. Notably, not all the brain Abeta peptides have identical solubility properties; whereas many of them are highly soluble in water-based physiologic solutions others require mild detergents or strong acids for extraction, suggesting their differential involvement in catabolic and fibrillogenic processes
— id: 136531, year: 2011, vol: 8, page: ?, stat: Journal Article,

The pseudokinase domain of JAK2 is a dual-specificity protein kinase that negatively regulates cytokine signaling
Ungureanu, Daniela; Wu, Jinhua; Pekkala, Tuija; Niranjan, Yashavanthi; Young, Clifford; Jensen, Ole N; Xu, Chong-Feng; Neubert, Thomas A; Skoda, Radek C; Hubbard, Stevan R; Silvennoinen, Olli
2011 ;18(9):971-976, Nature structural & molecular biology
Human JAK2 tyrosine kinase mediates signaling through numerous cytokine receptors. The JAK2 JH2 domain functions as a negative regulator and is presumed to be a catalytically inactive pseudokinase, but the mechanism(s) for its inhibition of JAK2 remains unknown. Mutations in JH2 lead to increased JAK2 activity, contributing to myeloproliferative neoplasms (MPNs). Here we show that JH2 is a dual-specificity protein kinase that phosphorylates two negative regulatory sites in JAK2: Ser523 and Tyr570. Inactivation of JH2 catalytic activity increased JAK2 basal activity and downstream signaling. Notably, different MPN mutations abrogated JH2 activity in cells, and in MPN (V617F) patient cells phosphorylation of Tyr570 was reduced, suggesting that loss of JH2 activity contributes to the pathogenesis of MPNs. These results identify the catalytic activity of JH2 as a previously unrecognized mechanism to control basal activity and signaling of JAK2
— id: 137017, year: 2011, vol: 18, page: 971, stat: Journal Article,

A Novel Transcription Complex That Selectively Modulates Apoptosis of Breast Cancer Cells through Regulation of FASTKD2
Yeung, Kay T; Das, Sharmistha; Zhang, Jin; Lomniczi, Alejandro; Ojeda, Sergio R; Xu, Chong-Feng; Neubert, Thomas A; Samuels, Herbert H
2011 Jun;31(11):2287-2298, Molecular & cellular biology
We previously reported that expression of NRIF3 (nuclear receptor interacting factor-3) rapidly and selectively leads to apoptosis of breast cancer cells. DIF-1 (also known as interferon regulatory factor-2 binding protein 2 [IRF-2BP2]), the cellular target of NRIF3, was identified as a transcriptional repressor, and DIF-1 knockdown leads to apoptosis of breast cancer cells but not other cell types. Here, we identify IRF-2BP1 and EAP1 (enhanced at puberty 1) as important components of the DIF-1 complex mediating both complex stability and transcriptional repression. This interaction of DIF-1, IRF-2BP1, and EAP1 occurs through the conserved C4 zinc fingers of these proteins. Microarray studies were carried out in breast cancer cell lines engineered to conditionally and rapidly increase the levels of the death domain (DD1) region of NRIF3. The DIF-1 complex was found to repress FASTKD2, a putative proapoptotic gene, in breast cancer cells and to bind to the FASTKD2 gene by chromatin immunoprecipitation. FASTKD2 knockdown prevents apoptosis of breast cancer cells from NRIF3 expression or DIF-1 knockdown, while expression of FASTKD2 leads to apoptosis of both breast and nonbreast cancer cells. Thus, regulation of FASTKD2 by NRIF3 and the DIF-1 complex acts as a novel death switch that selectively modulates apoptosis in breast cancer
— id: 132312, year: 2011, vol: 31, page: 2287, stat: Journal Article,

Study of Neurotrophin-3 Signaling in Primary Cultured Neurons using Multiplex Stable Isotope Labeling with Amino Acids in Cell Culture
Zhang, Guoan; Deinhardt, Katrin; Chao, Moses V; Neubert, Thomas A
2011 May 6;10(5):2546-2554, Journal of proteome research
Conventional stable isotope labeling with amino acids in cell culture (SILAC) requires extensive metabolic labeling of proteins and therefore is difficult to apply to cells that do not divide or are unstable in SILAC culture. Using two different sets of heavy amino acids for labeling allows for straightforward SILAC quantitation using partially labeled cells because the two cell populations are always equally labeled. Here we report the application of this labeling strategy to primary cultured neurons. We demonstrated that protein quantitation was not compromised by incomplete labeling of the neuronal proteins. We used this method to study neurotrophin-3 (NT-3) signaling in primary cultured neurons. Surprisingly our results indicate TrkB signaling is a major component of the signaling network induced by NT-3 in cortical neurons. In addition, involvement of proteins such as VAMP2, Scamp1, and Scamp3 suggests that NT-3 may lead to enhanced exocytosis of synaptic vesicles
— id: 132309, year: 2011, vol: 10, page: 2546, stat: Journal Article,

Comparison of three quantitative phosphoproteomic strategies to study receptor tyrosine kinase signaling
Zhang, Guoan; Neubert, Thomas A
2011 Dec 2;10(12):5454-5462, Journal of proteome research
There are three quantitative phosphoproteomic strategies most commonly used to study receptor tyrosine kinase (RTK) signaling. These strategies quantify changes in: (1) all three forms of phosphosites (phosphoserine, phosphothreonine and phosphotyrosine) following enrichment of phosphopeptides by titanium dioxide or immobilized metal affinity chromatography; (2) phosphotyrosine sites following anti- phosphotyrosine antibody enrichment of phosphotyrosine peptides; or (3) phosphotyrosine proteins and their binding partners following anti-phosphotyrosine protein immunoprecipitation. However, it is not clear from literature which strategy is more effective. In this study, we assessed the utility of these three phosphoproteomic strategies in RTK signaling studies by using EphB receptor signaling as an example. We used all three strategies with stable isotope labeling with amino acids in cell culture (SILAC) to compare changes in phosphoproteomes upon EphB receptor activation. We used bioinformatic analysis to compare results from the three analyses. Our results show that the three strategies provide complementary information about RTK pathways
— id: 145751, year: 2011, vol: 10, page: 5454, stat: Journal Article,

Recombinant derivatives of botulinum neurotoxin A engineered for trafficking studies and neuronal delivery
Band, Philip A; Blais, Steven; Neubert, Thomas A; Cardozo, Timothy J; Ichtchenko, Konstantin
2010 May;71(1):62-73, Protein expression & purification
Work from multiple laboratories has clarified how the structural domains of botulinum neurotoxin A (BoNT/A) disable neuronal exocytosis, but important questions remain unanswered. Because BoNT/A intoxication disables its own uptake, light chain (LC) does not accumulate in neurons at detectable levels. We have therefore designed, expressed and purified a series of BoNT/A atoxic derivatives (ad) that retain the wild type features required for native trafficking. BoNT/A1ad(ek) and BoNT/A1ad(tev) are full length derivatives rendered atoxic through double point mutations in the LC protease (E(224)>A; Y(366)>A). DeltaLC-peptide-BoNT/A(tev) and DeltaLC-GFP-BoNT/A(tev) are derivatives wherein the catalytic portion of the LC is replaced with a short peptide or with GFP plus the peptide. In all four derivatives, we have fused the S6 peptide sequence GDSLSWLLRLLN to the N-terminus of the proteins to enable site-specific attachment of cargo using Sfp phosphopantetheinyl transferase. Cargo can be attached in a manner that provides a homogeneous derivative population rather than a polydisperse mixture of singly and multiply-labeled molecular species. All four derivatives contain an introduced cleavage site for conversion into disulfide-bonded heterodimers. These constructs were expressed in a baculovirus system and the proteins were secreted into culture medium and purified to homogeneity in yields ranging from 1 to 30 mg per liter. These derivatives provide unique tools to study toxin trafficking in vivo, and to assess how the structure of cargo linked to the heavy chain (HC) influences delivery to the neuronal cytosol. Moreover, they create the potential to engineer BoNT-based molecular vehicles that can target therapeutic agents to the neuronal cytoplasm
— id: 107925, year: 2010, vol: 71, page: 62, stat: Journal Article,

Canonical and alternate functions of the microRNA biogenesis machinery
Chong, Mark M W; Zhang, Guoan; Cheloufi, Sihem; Neubert, Thomas A; Hannon, Gregory J; Littman, Dan R
2010 Sep 1;24(17):1951-1960, Genes & development
The canonical microRNA (miRNA) biogenesis pathway requires two RNaseIII enzymes: Drosha and Dicer. To understand their functions in mammals in vivo, we engineered mice with germline or tissue-specific inactivation of the genes encoding these two proteins. Changes in proteomic and transcriptional profiles that were shared in Dicer- and Drosha-deficient mice confirmed the requirement for both enzymes in canonical miRNA biogenesis. However, deficiency in Drosha or Dicer did not always result in identical phenotypes, suggesting additional functions. We found that, in early-stage thymocytes, Drosha recognizes and directly cleaves many protein-coding messenger RNAs (mRNAs) with secondary stem-loop structures. In addition, we identified a subset of miRNAs generated by a Dicer-dependent but Drosha-independent mechanism. These were distinct from previously described mirtrons. Thus, in mammalian cells, Dicer is required for the biogenesis of multiple classes of miRNAs. Together, these findings extend the range of function of RNaseIII enzymes beyond canonical miRNA biogenesis, and help explain the nonoverlapping phenotypes caused by Drosha and Dicer deficiency
— id: 112041, year: 2010, vol: 24, page: 1951, stat: Journal Article,

Dok-7 regulates neuromuscular synapse formation by recruiting Crk and Crk-L
Hallock, Peter T; Xu, Chong-Feng; Park, Tae-Ju; Neubert, Thomas A; Curran, Tom; Burden, Steven J
2010 Nov 1;24(21):2451-2461, Genes & development
Agrin, released by motor neurons, promotes neuromuscular synapse formation by stimulating MuSK, a receptor tyrosine kinase expressed in skeletal muscle. Phosphorylated MuSK recruits docking protein-7 (Dok-7), an adaptor protein that is expressed selectively in muscle. In the absence of Dok-7, neuromuscular synapses fail to form, and mutations that impair Dok-7 are a major cause of congenital myasthenia in humans. How Dok-7 stimulates synaptic differentiation is poorly understood. Once recruited to MuSK, Dok-7 directly stimulates MuSK kinase activity. This unusual activity of an adapter protein is mediated by the N-terminal region of Dok-7, whereas most mutations that cause congenital myasthenia truncate the C-terminal domain. Here, we demonstrate that Dok-7 also functions downstream from MuSK, and we identify the proteins that are recruited to the C-terminal domain of Dok-7. We show that Agrin stimulates phosphorylation of two tyrosine residues in the C-terminal domain of Dok-7, which leads to recruitment of two adapter proteins: Crk and Crk-L. Furthermore, we show that selective inactivation of Crk and Crk-L in skeletal muscle leads to severe defects in neuromuscular synapses in vivo, revealing a critical role for Crk and Crk-L downstream from Dok-7 in presynaptic and postsynaptic differentiation
— id: 114184, year: 2010, vol: 24, page: 2451, stat: Journal Article,

The matrix peptide exporter HAF-1 signals a mitochondrial UPR by activating the transcription factor ZC376.7 in C. elegans
Haynes, Cole M; Yang, Yun; Blais, Steven P; Neubert, Thomas A; Ron, David
2010 Feb 26;37(4):529-540, Molecular cell
Genetic analyses previously implicated the matrix-localized protease ClpP in signaling the stress of protein misfolding in the mitochondrial matrix to activate nuclear-encoded mitochondrial chaperone genes in C. elegans (UPR(mt)). Here, we report that haf-1, a gene encoding a mitochondria-localized ATP-binding cassette protein, is required for signaling within the UPR(mt) and for coping with misfolded protein stress. Peptide efflux from isolated mitochondria was ATP dependent and required HAF-1 and the protease ClpP. Defective UPR(mt) signaling in the haf-1-deleted worms was associated with failure of the bZIP protein, ZC376.7, to localize to nuclei in worms with perturbed mitochondrial protein folding, whereas zc376.7(RNAi) strongly inhibited the UPR(mt). These observations suggest a simple model whereby perturbation of the protein-folding environment in the mitochondrial matrix promotes ClpP-mediated generation of peptides whose haf-1-dependent export from the matrix contributes to UPR(mt) signaling across the mitochondrial inner membrane
— id: 107928, year: 2010, vol: 37, page: 529, stat: Journal Article,

Matrix metalloproteinase 2 (MMP-2) degrades soluble vasculotropic amyloid-beta E22Q and L34V mutants, delaying their toxicity for human brain microvascular endothelial cells
Hernandez-Guillamon, Mar; Mawhirt, Stephanie; Fossati, Silvia; Blais, Steven; Pares, Mireia; Penalba, Anna; Boada, Merce; Couraud, Pierre-Olivier; Neubert, Thomas A; Montaner, Joan; Ghiso, Jorge; Rostagno, Agueda
2010 Aug 27;285(35):27144-27158, Journal of biological chemistry
Patients carrying mutations within the amyloid-beta (Abeta) sequence develop severe early-onset cerebral amyloid angiopathy with some of the related variants manifesting primarily with hemorrhagic phenotypes. Matrix metalloproteases (MMPs) are typically associated with blood brain barrier disruption and hemorrhagic transformations after ischemic stroke. However, their contribution to cerebral amyloid angiopathy-related hemorrhage remains unclear. Human brain endothelial cells challenged with Abeta synthetic homologues containing mutations known to be associated in vivo with hemorrhagic manifestations (AbetaE22Q and AbetaL34V) showed enhanced production and activation of MMP-2, evaluated via Multiplex MMP antibody arrays, gel zymography, and Western blot, which in turn proteolytically cleaved in situ the Abeta peptides. Immunoprecipitation followed by mass spectrometry analysis highlighted the generation of specific C-terminal proteolytic fragments, in particular the accumulation of Abeta-(1-16), a result validated in vitro with recombinant MMP-2 and quantitatively evaluated using deuterium-labeled internal standards. Silencing MMP-2 gene expression resulted in reduced Abeta degradation and enhanced apoptosis. Secretion and activation of MMP-2 as well as susceptibility of the Abeta peptides to MMP-2 degradation were dependent on the peptide conformation, with fibrillar elements of AbetaE22Q exhibiting negligible effects. Our results indicate that MMP-2 release and activation differentially degrades Abeta species, delaying their toxicity for endothelial cells. However, taking into consideration MMP ability to degrade basement membrane components, these protective effects might also undesirably compromise blood brain barrier integrity and precipitate a hemorrhagic phenotype
— id: 112035, year: 2010, vol: 285, page: 27144, stat: Journal Article,

Phosphorylation of the PRC2 component Ezh2 is cell cycle-regulated and up-regulates its binding to ncRNA
Kaneko, Syuzo; Li, Gang; Son, Jinsook; Xu, Chong-Feng; Margueron, Raphael; Neubert, Thomas A; Reinberg, Danny
2010 Dec 1;24(23):2615-2620, Genes & development
Ezh2 functions as a histone H3 Lys 27 (H3K27) methyltransferase when comprising the Polycomb-Repressive Complex 2 (PRC2). Trimethylation of H3K27 (H3K27me3) correlates with transcriptionally repressed chromatin. The means by which PRC2 targets specific chromatin regions is currently unclear, but noncoding RNAs (ncRNAs) have been shown to interact with PRC2 and may facilitate its recruitment to some target genes. Here we show that Ezh2 interacts with HOTAIR and Xist. Ezh2 is phosphorylated by cyclin-dependent kinase 1 (CDK1) at threonine residues 345 and 487 in a cell cycle-dependent manner. A phospho-mimic at residue 345 increased HOTAIR ncRNA binding to Ezh2, while the phospho-mimic at residue 487 was ineffectual. An Ezh2 domain comprising T345 was found to be important for binding to HOTAIR and the 5' end of Xist
— id: 114862, year: 2010, vol: 24, page: 2615, stat: Journal Article,

Catabolism of Alzheimer's amyloid-beta: Implications for brain clearance and plaque deposition
McIntee F.L.; Neubert T.; Blais S.; Ghiso J.
2010 ;3(2):S47-S47, Clinical & Translational Science
OBJECTIVES: Alzheimer's disease (AD) is the leading cause of dementia and the most common form of amyloidosis in humans. Extensive extracellular deposition of amyloid-beta (Abeta), a 40-42 amino acid degradation product of APP, is considered a hallmark feature of AD. Our attention is focused on the highly heterogeneous biochemical nature of the brain Abeta species. METHODS AND POPULATION: We have fractionated water-soluble, detergent-soluble and formic acid-solube Abeta species from transgenic mouse models of amyloid deposition and AD cases. Subsequently, we applied a combination of biochemical techniques including immunoprecipitation followed by identification of Abeta fragments with a novel highly sensitive matrixassisted laser desorption/ionization time-of-flight mass spectrometry technique. RESULTS: Our biochemical data on the Abeta species present in sporadic and familial AD cases and in transgenic mouse models highlight the presence of similar N- and C-terminally truncated fragments-likely reflecting the ability of multiple proteases to degrade Abeta in situ- and several post-translational modifications with still unclear roles in the amyloidogenesis mechanism. Notably, not all the brain Abeta peptides have identical solubility properties. SIGNIFICANCE OF STUDY: In view of these findings and the growing evidence that an imbalance between Abeta production and clearance plays a major role in the process of Abeta deposition, we hypothesize that certain truncated and post-translationally modified Abeta fragments have a distinct and opposite role in clearance and fibrillization mechanisms and that the spectrum and abundance of these species vary according to the magnitude of the amyloid load
— id: 111407, year: 2010, vol: 3, page: S47, stat: Journal Article,

Super-SILAC for tumors and tissues
Neubert, TA; Tempst, P
2010 MAY ;7(5):361-362, Nature methods
— id: 109681, year: 2010, vol: 7, page: 361, stat: Journal Article,

Interlaboratory study characterizing a yeast performance standard for benchmarking LC-MS platform performance
Paulovich, Amanda G; Billheimer, Dean; Ham, Amy-Joan L; Vega-Montoto, Lorenzo; Rudnick, Paul A; Tabb, David L; Wang, Pei; Blackman, Ronald K; Bunk, David M; Cardasis, Helene L; Clauser, Karl R; Kinsinger, Christopher R; Schilling, Birgit; Tegeler, Tony J; Variyath, Asokan Mulayath; Wang, Mu; Whiteaker, Jeffrey R; Zimmerman, Lisa J; Fenyo, David; Carr, Steven A; Fisher, Susan J; Gibson, Bradford W; Mesri, Mehdi; Neubert, Thomas A; Regnier, Fred E; Rodriguez, Henry; Spiegelman, Cliff; Stein, Stephen E; Tempst, Paul; Liebler, Daniel C
2010 Feb;9(2):242-254, Molecular & cellular proteomics
Optimal performance of LC-MS/MS platforms is critical to generating high quality proteomics data. Although individual laboratories have developed quality control samples, there is no widely available performance standard of biological complexity (and associated reference data sets) for benchmarking of platform performance for analysis of complex biological proteomes across different laboratories in the community. Individual preparations of the yeast Saccharomyces cerevisiae proteome have been used extensively by laboratories in the proteomics community to characterize LC-MS platform performance. The yeast proteome is uniquely attractive as a performance standard because it is the most extensively characterized complex biological proteome and the only one associated with several large scale studies estimating the abundance of all detectable proteins. In this study, we describe a standard operating protocol for large scale production of the yeast performance standard and offer aliquots to the community through the National Institute of Standards and Technology where the yeast proteome is under development as a certified reference material to meet the long term needs of the community. Using a series of metrics that characterize LC-MS performance, we provide a reference data set demonstrating typical performance of commonly used ion trap instrument platforms in expert laboratories; the results provide a basis for laboratories to benchmark their own performance, to improve upon current methods, and to evaluate new technologies. Additionally, we demonstrate how the yeast reference, spiked with human proteins, can be used to benchmark the power of proteomics platforms for detection of differentially expressed proteins at different levels of concentration in a complex matrix, thereby providing a metric to evaluate and minimize pre-analytical and analytical variation in comparative proteomics experiments
— id: 114127, year: 2010, vol: 9, page: 242, stat: Journal Article,

Myristoylation of the dual-specificity phosphatase c-JUN N-terminal kinase (JNK) stimulatory phosphatase 1 is necessary for its activation of JNK signaling and apoptosis
Schwertassek, U; Buckley, DA; Xu, CF; Lindsay, AJ; McCaffrey, MW; Neubert, TA; Tonks, NK
2010 JUN ;277(11):2463-2473, FEBS journal
Activation of the c-JUN N-terminal kinase (JNK) pathway is implicated in a number of important physiological processes, from embryonic morphogenesis to cell survival and apoptosis. JNK stimulatory phosphatase 1 (JSP1) is a member of the dual-specificity phosphatase subfamily of protein tyrosine phosphatases. In contrast to other dual-specificity phosphatases that catalyze the inactivation of mitogen-activated protein kinases, expression of JSP1 activates JNK-mediated signaling. JSP1 and its relative DUSP15 are unique among members of the protein tyrosine phosphatase family in that they contain a potential myristoylation site at the N-terminus (MGNGMXK). In this study, we investigated whether JSP1 was myristoylated and examined the functional consequences of myristoylation. Using mass spectrometry, we showed that wild-type JSP1, but not a JSP1 mutant in which Gly2 was mutated to Ala (JSP1-G2A), was myristoylated in cells. Although JSP1 maintained intrinsic phosphatase activity in the absence of myristoylation, the subcellular localization of the enzyme was altered. Compared with the wild type, the ability of nonmyristoylated JSP1 to induce JNK activation and phosphorylation of the transcription factor c-JUN was attenuated. Upon expression of wild-type JSP1, a subpopulation of cells, with the highest levels of the phosphatase, was induced to float off the dish and undergo apoptosis. In contrast, cells expressing similar levels of JSP1-G2A remained attached, further highlighting that the myristoylation mutant was functionally compromised
— id: 109833, year: 2010, vol: 277, page: 2463, stat: Journal Article,

Repeatability and reproducibility in proteomic identifications by liquid chromatography-tandem mass spectrometry
Tabb, David L; Vega-Montoto, Lorenzo; Rudnick, Paul A; Variyath, Asokan Mulayath; Ham, Amy-Joan L; Bunk, David M; Kilpatrick, Lisa E; Billheimer, Dean D; Blackman, Ronald K; Cardasis, Helene L; Carr, Steven A; Clauser, Karl R; Jaffe, Jacob D; Kowalski, Kevin A; Neubert, Thomas A; Regnier, Fred E; Schilling, Birgit; Tegeler, Tony J; Wang, Mu; Wang, Pei; Whiteaker, Jeffrey R; Zimmerman, Lisa J; Fisher, Susan J; Gibson, Bradford W; Kinsinger, Christopher R; Mesri, Mehdi; Rodriguez, Henry; Stein, Stephen E; Tempst, Paul; Paulovich, Amanda G; Liebler, Daniel C; Spiegelman, Cliff
2010 Feb 5;9(2):761-776, Journal of proteome research
The complexity of proteomic instrumentation for LC-MS/MS introduces many possible sources of variability. Data-dependent sampling of peptides constitutes a stochastic element at the heart of discovery proteomics. Although this variation impacts the identification of peptides, proteomic identifications are far from completely random. In this study, we analyzed interlaboratory data sets from the NCI Clinical Proteomic Technology Assessment for Cancer to examine repeatability and reproducibility in peptide and protein identifications. Included data spanned 144 LC-MS/MS experiments on four Thermo LTQ and four Orbitrap instruments. Samples included yeast lysate, the NCI-20 defined dynamic range protein mix, and the Sigma UPS 1 defined equimolar protein mix. Some of our findings reinforced conventional wisdom, such as repeatability and reproducibility being higher for proteins than for peptides. Most lessons from the data, however, were more subtle. Orbitraps proved capable of higher repeatability and reproducibility, but aberrant performance occasionally erased these gains. Even the simplest protein digestions yielded more peptide ions than LC-MS/MS could identify during a single experiment. We observed that peptide lists from pairs of technical replicates overlapped by 35-60%, giving a range for peptide-level repeatability in these experiments. Sample complexity did not appear to affect peptide identification repeatability, even as numbers of identified spectra changed by an order of magnitude. Statistical analysis of protein spectral counts revealed greater stability across technical replicates for Orbitraps, making them superior to LTQ instruments for biomarker candidate discovery. The most repeatable peptides were those corresponding to conventional tryptic cleavage sites, those that produced intense MS signals, and those that resulted from proteins generating many distinct peptides. Reproducibility among different instruments of the same type lagged behind repeatability of technical replicates on a single instrument by several percent. These findings reinforce the importance of evaluating repeatability as a fundamental characteristic of analytical technologies
— id: 134986, year: 2010, vol: 9, page: 761, stat: Journal Article,

Iowa variant of familial Alzheimer's disease: accumulation of posttranslationally modified AbetaD23N in parenchymal and cerebrovascular amyloid deposits
Tomidokoro, Yasushi; Rostagno, Agueda; Neubert, Thomas A; Lu, Yun; Rebeck, G William; Frangione, Blas; Greenberg, Steven M; Ghiso, Jorge
2010 Apr;176(4):1841-1854, American journal of pathology
Mutations within the amyloid-beta (Abeta) sequence, especially those clustered at residues 21-23, which are linked to early onset familial Alzheimer's disease (AD), are primarily associated with cerebral amyloid angiopathy (CAA). The basis for this predominant vascular amyloid burden and the differential clinical phenotypes of cerebral hemorrhage/stroke in some patients and dementia in others remain unknown. The AbetaD23N Iowa mutation is associated with progressive AD-like dementia, often without clinically manifested intracerebral hemorrhage. Neuropathologically, the disease is characterized by predominant preamyloid deposits, severe CAA, and abundant neurofibrillary tangles in the presence of remarkably few mature plaques. Biochemical analyses using a combination of immunoprecipitation, mass spectrometry, amino acid sequence, and Western blot analysis performed after sequential tissue extractions to separately isolate soluble components, preamyloid, and fibrillar amyloid species indicated that the Iowa deposits are complex mixtures of mutated and nonmutated Abeta molecules. These molecules exhibited various degrees of solubility, were highly heterogeneous at both the N- and C-termini, and showed partial aspartate isomerization at positions 1, 7, and 23. This collection of Abeta species-the Iowa brain Abeta peptidome-contained clear imprints of amyloid clearance mechanisms yet highlighted the unique neuropathological features shared by a non-Abeta cerebral amyloidosis, familial Danish dementia, in which neurofibrillary tangles coexist with extensive pre-amyloid deposition in the virtual absence of fibrillar lesions. These data therefore challenge the importance of neuritic plaques as the sole contributors for the development of dementia
— id: 108922, year: 2010, vol: 176, page: 1841, stat: Journal Article,

Overview of peptide and protein analysis by mass spectrometry
Zhang G.; Annan R.S.; Carr S.A.; Neubert T.A.
2010 ;Suppl 62:?-? #16.1, Current protocols in protein science
Mass spectrometry is an indispensable tool for peptide and protein analysis owing to its speed, sensitivity, and versatility. It can be used to determine amino acid sequences of peptides, and to characterize a wide variety of post-translational modifications such as phosphorylation and glycosylation. Mass spectrometry can also be used to determine absolute and relative protein quantities, and can identify and quantify thousands of proteins from complex samples, which makes it an extremely powerful tool for systems biology studies. The main goals of this unit are to familiarize peptide and protein chemists and biologists with the types of mass spectrometers that are appropriate for the majority of their analytical needs, to describe the kinds of experiments that can be performed with these instruments on a routine basis, and to discuss the kinds of information that these experiments provide. 2010 by John Wiley & Sons, Inc
— id: 131982, year: 2010, vol: Suppl 62, page: ?, stat: Journal Article,

Overview of peptide and protein analysis by mass spectrometry
Zhang, Guoan; Annan, Roland S; Carr, Steven A; Neubert, Thomas A
2010 Nov;Chapter 16:Unit16.1-Unit16.1, Current protocols in protein science
Mass spectrometry is an indispensable tool for peptide and protein analysis owing to its speed, sensitivity, and versatility. It can be used to determine amino acid sequences of peptides, and to characterize a wide variety of post-translational modifications such as phosphorylation and glycosylation. Mass spectrometry can also be used to determine absolute and relative protein quantities, and can identify and quantify thousands of proteins from complex samples, which makes it an extremely powerful tool for systems biology studies. The main goals of this unit are to familiarize peptide and protein chemists and biologists with the types of mass spectrometers that are appropriate for the majority of their analytical needs, to describe the kinds of experiments that can be performed with these instruments on a routine basis, and to discuss the kinds of information that these experiments provide
— id: 114845, year: 2010, vol: Chapter 16, page: Unit16.1, stat: Journal Article,

Protein quantitation using mass spectrometry
Zhang, Guoan; Ueberheide, Beatrix M; Waldemarson, Sofia; Myung, Sunnie; Molloy, Kelly; Eriksson, Jan; Chait, Brian T; Neubert, Thomas A; Fenyo, David
2010 ;673:211-222, Methods in molecular biology
Mass spectrometry is a method of choice for quantifying low-abundance proteins and peptides in many biological studies. Here, we describe a range of computational aspects of protein and peptide quantitation, including methods for finding and integrating mass spectrometric peptide peaks, and detecting interference to obtain a robust measure of the amount of proteins present in samples
— id: 112434, year: 2010, vol: 673, page: 211, stat: Journal Article,

Oxidative protein folding by an endoplasmic reticulum-localized peroxiredoxin
Zito, Ester; Melo, Eduardo Pinho; Yang, Yun; Wahlander, Asa; Neubert, Thomas A; Ron, David
2010 Dec 10;40(5):787-797, Molecular cell
Endoplasmic reticulum (ER) oxidation 1 (ERO1) transfers disulfides to protein disulfide isomerase (PDI) and is essential for oxidative protein folding in simple eukaryotes such as yeast and worms. Surprisingly, ERO1-deficient mammalian cells exhibit only a modest delay in disulfide bond formation. To identify ERO1-independent pathways to disulfide bond formation, we purified PDI oxidants with a trapping mutant of PDI. Peroxiredoxin IV (PRDX4) stood out in this list, as the related cytosolic peroxiredoxins are known to form disulfides in the presence of hydroperoxides. Mouse embryo fibroblasts lacking ERO1 were intolerant of PRDX4 knockdown. Introduction of wild-type mammalian PRDX4 into the ER rescued the temperature-sensitive phenotype of an ero1 yeast mutation. In the presence of an H(2)O(2)-generating system, purified PRDX4 oxidized PDI and reconstituted oxidative folding of RNase A. These observations implicate ER-localized PRDX4 in a previously unanticipated, parallel, ERO1-independent pathway that couples hydroperoxide production to oxidative protein folding in mammalian cells
— id: 115430, year: 2010, vol: 40, page: 787, stat: Journal Article,

Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma
Addona, TA; Abbatiello, SE; Schilling, B; Skates, SJ; Mani, DR; Bunk, DM; Spiegelman, CH; Zimmerman, LJ; Ham, AJL; Keshishian, H; Hall, SC; Allen, S; Blackman, RK; Borchers, CH; Buck, C; Cardasis, HL; Cusack, MP; Dodder, NG; Gibson, BW; Held, JM; Hiltke, T; Jackson, A; Johansen, EB; Kinsinger, CR; Li, J; Mesri, M; Neubert, TA; Niles, RK; Pulsipher, TC; Ransohoff, D; Rodriguez, H; Rudnick, PA; Smith, D; Tabb, DL; Tegeler, TJ; Variyath, AM; Vega-Montoto, LJ; Wahlander, A; Waldemarson, S; Wang, M; Whiteaker, JR; Zhao, L; Anderson, NL; Fisher, SJ; Liebler, DC; Paulovich, AG; Regnier, FE; Tempst, P; Carr, SA
2009 JUL ;27(7):633-U85, Nature biotechnology
Verification of candidate biomarkers relies upon specific, quantitative assays optimized for selective detection of target proteins, and is increasingly viewed as a critical step in the discovery pipeline that bridges unbiased biomarker discovery to preclinical validation. Although individual laboratories have demonstrated that multiple reaction monitoring (MRM) coupled with isotope dilution mass spectrometry can quantify candidate protein biomarkers in plasma, reproducibility and transferability of these assays between laboratories have not been demonstrated. We describe a multilaboratory study to assess reproducibility, recovery, linear dynamic range and limits of detection and quantification of multiplexed, MRM-based assays, conducted by NCI-CPTAC. Using common materials and standardized protocols, we demonstrate that these assays can be highly reproducible within and across laboratories and instrument platforms, and are sensitive to low lg/ml protein concentrations in unfractionated plasma. We provide data and benchmarks against which individual laboratories can compare their performance and evaluate new technologies for biomarker verification in plasma
— id: 101086, year: 2009, vol: 27, page: 633, stat: Journal Article,

Identification and characterization of a novel nuclear protein complex Involved In nuclear hormone receptor-mediated gene regulation
Garapaty, Shivani; Xu, Chong-Feng; Trojer, Patrick; Mahajan, Muktar A; Neubert, Thomas A; Samuels, Herbert H
2009 Mar 20;284(12):7542-7552, Journal of biological chemistry
NRC/NCoA6 plays an important role in mediating the effects of ligand-bound nuclear hormone receptors as well as other transcription factors. NRC interacting factor 1 (NIF-1) was cloned as a novel factor that interacts in vivo with NRC. Although NIF-1 does not directly interact with nuclear hormone receptors, it enhances activation by nuclear hormone receptors presumably through its interaction with NRC. To further understand the cellular and biological function of NIF-1, we identified NIF-1 associated proteins by in-solution proteolysis followed by mass spectrometry. The identified components revealed factors involved in histone methylation and cell cycle control and include Ash2L, RbBP5, WDR5, HCF-1, DBC-1, and EMSY. Although the NIF-1 complex contains Ash2L, RbBP5, and WDR5 suggesting that the complex might methylate histone H3-Lys4, we found that the complex contains a H3 methyltransferase activity that modifies a residue other than H3-Lys 4. The identified components form at least two distinct sized NIF-1 complexes. DBC-1 and EMSY were identified as integral components of a ~1.5 MDa NIF-1 complex and were found to play an important role in the regulation of nuclear receptor-mediated transcription. Stimulation of the Sox9 and HoxA1 genes by retinoic acid receptor-a was found to require both DBC-1 and EMSY in addition to NIF-1 for maximal transcriptional activation. Interestingly, NRC was not identified as a component of the NIF-1 complex, suggesting that NIF-1 and NRC do not exist as stable in vitro purified complexes although the separate NIF-1 and NRC complexes appear to functionally interact in the cell
— id: 95312, year: 2009, vol: 284, page: 7542, stat: Journal Article,

Crystal structure of a fibroblast growth factor homologous factor (FHF) defines a conserved surface on FHFs for binding and modulation of voltage-gated sodium channels
Goetz, Regina; Dover, Katarzyna; Laezza, Fernanda; Shtraizent, Nataly; Huang, Xiao; Tchetchik, Dafna; Eliseenkova, Anna V; Xu, Chong-Feng; Neubert, Thomas A; Ornitz, David M; Goldfarb, Mitchell; Mohammadi, Moosa
2009 Jun 26;284(26):17883-17896, Journal of biological chemistry
Voltage-gated sodium channels (Nav) produce sodium currents that underlie the initiation and propagation of action potentials in nerve and muscle cells. Fibroblast growth factor homologous factors (FHFs) bind to the intracellular C-terminal region of the Nav alpha subunit to modulate fast inactivation of the channel. In this study we solved the crystal structure of a 149-residue-long fragment of human FHF2A which unveils the structural features of the homology core domain of all 10 human FHF isoforms. Through analysis of crystal packing contacts and site-directed mutagenesis experiments we identified a conserved surface on the FHF core domain that mediates channel binding in vitro and in vivo. Mutations at this channel binding surface impaired the ability of FHFs to co-localize with Navs at the axon initial segment of hippocampal neurons. The mutations also disabled FHF modulation of voltage-dependent fast inactivation of sodium channels in neuronal cells. Based on our data, we propose that FHFs constitute auxiliary subunits for Navs
— id: 100603, year: 2009, vol: 284, page: 17883, stat: Journal Article,

Homodimerization controls the fibroblast growth factor 9 subfamily's receptor binding and heparan sulfate-dependent diffusion in the extracellular matrix
Kalinina, Juliya; Byron, Sara A; Makarenkova, Helen P; Olsen, Shaun K; Eliseenkova, Anna V; Larochelle, William J; Dhanabal, Mohanraj; Blais, Steven; Ornitz, David M; Day, Loren A; Neubert, Thomas A; Pollock, Pamela M; Mohammadi, Moosa
2009 Sep;29(17):4663-4678, Mol Cell Biol
Uncontrolled fibroblast growth factor (FGF) signaling can lead to human diseases, necessitating multiple layers of self-regulatory control mechanisms to keep its activity in check. Herein, we demonstrate that FGF9 and FGF20 ligands undergo a reversible homodimerization, occluding their key receptor binding sites. To test the role of dimerization in ligand autoinhibition, we introduced structure-based mutations into the dimer interfaces of FGF9 and FGF20. The mutations weakened the ability of the ligands to dimerize, effectively increasing the concentrations of monomeric ligands capable of binding and activating their cognate FGF receptor in vitro and in living cells. Interestingly, the monomeric ligands exhibit reduced heparin binding, resulting in their increased radii of heparan sulfate-dependent diffusion and biologic action, as evidenced by the wider dilation area of ex vivo lung cultures in response to implanted mutant FGF9-loaded beads. Hence, our data demonstrate that homodimerization autoregulates FGF9 and FGF20's receptor binding and concentration gradients in the extracellular matrix. Our study is the first to implicate ligand dimerization as an autoregulatory mechanism for growth factor bioactivity and sets the stage for engineering modified FGF9 subfamily ligands, with desired activity for use in both basic and translational research.
— id: 156085, year: 2009, vol: 29, page: 4663, stat: Journal Article,

Dissociation of the subunits of the calcium-independent receptor of alpha-latrotoxin as a result of two-step proteolysis
Krasnoperov, Valery; Deyev, Igor E; Serova, Oxana V; Xu, Chongfeng; Lu, Yun; Buryanovsky, Leonid; Gabibov, Alexander G; Neubert, Thomas A; Petrenko, Alexander G
2009 Apr 14;48(14):3230-3238, Biochemistry
CIRL (the calcium-independent receptor of alpha-latrotoxin), a neuronal cell surface receptor implicated in the regulation of exocytosis, is a member of the GPS family of chimeric cell adhesion/G protein-coupled receptors. The predominant form of CIRL is a membrane-bound complex of two subunits, p120 and p85. Extracellularly oriented p120 contains hydrophilic cell adhesion domains, whereas p85 is a heptahelical membrane protein. Both subunits are encoded by the same gene and represent products of intracellular proteolytic processing of the CIRL precursor. In this study, we demonstrate that a soluble form of CIRL also exists in vitro and in vivo. It results from the further cleavage of CIRL by a second protease. The site of the second cleavage is located in the short N-terminal extracellular tail of p85, between the GPS domain and the first transmembrane segment of CIRL. Thus, the soluble form of CIRL represents a complex of p120 noncovalently bound to a 15 amino acid residue N-terminal peptide fragment of p85. We have previously shown that mutations of CIRL in the GPS domain inhibit intracellular proteolytic processing and also result in the absence of the receptors from the cell surface. Our current data suggest that although CIRL trafficking to the cell membrane is impaired by mutations in the GPS region, it is not blocked completely. However, at the cell surface, the noncleaved mutants are preferentially targeted by the second protease that sheds the extracellular subunit. Therefore, the two-step proteolytic processing may represent a regulatory mechanism that controls cell surface expression of membrane-bound and soluble forms of CIRL
— id: 98778, year: 2009, vol: 48, page: 3230, stat: Journal Article,

The target of the NSD family of histone lysine methyltransferases depends on the nature of the substrate
Li, Yan; Trojer, Patrick; Xu, Chong-Feng; Cheung, Peggie; Kuo, Alex; Drury, William J 3rd; Qiao, Qi; Neubert, Thomas A; Xu, Rui-Ming; Gozani, Or; Reinberg, Danny
2009 Dec 4;284(49):34283-34295, Journal of biological chemistry
The NSD (nuclear receptor SET domain-containing) family of histone lysine methyltransferases is a critical participant in chromatin integrity as evidenced by the number of human diseases associated with the aberrant expression of its family members. Yet, the specific targets of these enzymes are not clear, with marked discrepancies being reported in the literature. We demonstrate that NSD2 can exhibit disparate target preferences based on the nature of the substrate provided. The NSD2 complex purified from human cells and recombinant NSD2 both exhibit specific targeting of histone H3 lysine 36 (H3K36) when provided with nucleosome substrates, but histone H4 lysine 44 is the primary target in the case of octamer substrates, irrespective of the histones being native or recombinant. This disparity is negated when NSD2 is presented with octamer targets in conjunction with short single- or double-stranded DNA. Although the octamers cannot form nucleosomes, the target is nonetheless nucleosome-specific as is the product, dimethylated H3K36. This study clarifies in part the previous discrepancies reported with respect to NSD targets. We propose that DNA acts as an allosteric effector of NSD2 such that H3K36 becomes the preferred target
— id: 105498, year: 2009, vol: 284, page: 34283, stat: Journal Article,

On-membrane tryptic digestion of proteins for mass spectrometry analysis
Luque-Garcia, Jose L; Neubert, Thomas A
2009 ;536:331-341, Methods in molecular biology
Identification of proteins and characterization of posttranslational modifications are crucial steps for many biological, biochemical, and biomedical studies, and mass spectrometry has become the method of choice for these analyses. Here we describe two methods for the on-membrane digestion of proteins electroblotted onto nitrocellulose membranes prior to analysis by mass spectrometry. These on-membrane methods take approximately half the time of in-gel digestion and provide better digestion efficiency, due to the better accessibility of the protease to the proteins adsorbed onto the nitrocellulose, and better protein sequence coverage, especially for membrane proteins where large and hydrophobic peptides are commonly present
— id: 99320, year: 2009, vol: 536, page: 331, stat: Journal Article,

Characterization of novel oxidation products of cysteine in an active site motif peptide of PTP1B
Shetty, Vivekananda; Neubert, Thomas A
2009 Aug;20(8):1540-1548, Journal of the American Society for Mass Spectrometry
We investigated the formation of hydroxyl radical (OH(*)) and H(2)O(2) mediated oxidation products of a synthetic peptide, HCSAGIGRS, which is an active site sequence motif of protein tyrosine phosphatase 1B (PTP1B). We determined that a novel cysteine sulfinamide HC[S(O)N]SAGIGRS is produced in the oxidation reaction by Fenton reagents (Fe(+2)/H(2)O(2)) as well as by H(2)O(2). These products were characterized by tandem mass spectrometry experiments on both singly and doubly charged precursor ions. MS(3) experiments using an ion trap instrument as well as LC-MS/MS experiments using a quadrupole time-of-flight (Q-TOF) instrument demonstrated that HC[S(O)N]SAGIGRS is not a water loss product of cysteine sulfinic acid [HC(SO(2)H)SAGIGRS]. We also obtained data from tandem mass spectrometry experiments that provided evidence for the existence of stable cysteine sulfenic acid [HC(SOH)SAGIGRS] in solution. A mechanism for the formation of the cysteine sulfinamide product is proposed based on the above experimental results. The preparation and identification of cysteine sulfinamide in this study may provide insight into the mechanism of both OH(*) and H(2)O(2) induced oxidation reactions of protein tyrosine phosphatases
— id: 101283, year: 2009, vol: 20, page: 1540, stat: Journal Article,

Thioredoxin-related Protein 32 Is an Arsenite-regulated Thiol Reductase of the Proteasome 19 S Particle
Wiseman, R Luke; Chin, King-Tung; Haynes, Cole M; Stanhill, Ariel; Xu, Chong-Feng; Roguev, Assen; Krogan, Nevan J; Neubert, Thomas A; Ron, David
2009 May 29;284(22):15233-15245, Journal of biological chemistry
Perturbation of the cytoplasmic protein folding environment by exposure to oxidative stress-inducing As(III)-containing compounds challenges the ubiquitin-proteasome system. Here we report on mass spectrometric analysis of As(III)-induced changes in the proteasome's composition in samples prepared by stable isotope labeling with amino acids in cell culture, using mammalian cells in which TRP32 (thioredoxin-related protein of 32 kDa; also referred to as TXNL1) was identified as a novel subunit of the 26 S proteasome. Quantitative genetic interaction mapping, using the epistatic miniarray profiling approach, identified a functional connection between TRP32 and the proteasome. Deletion of txl1, the Schizosaccharomyces pombe homolog of TRP32, results in a slow growth phenotype when combined with deletion of cut8, a gene required for normal proteasome localization. Deletion analysis in vivo, chemical cross-linking, and manipulation of the ATP concentration in vitro during proteasome immunopurification revealed that the C-terminal domain of mammalian TRP32 binds the 19 S regulatory particle in proximity to the proteasome substrate binding site. Thiol modification with polyethylene glycol-maleimide showed disulfide bond formation at the active site of TRP32 in cells exposed to As(III). Pulse-chase labeling showed that TRP32 is a stable protein whose half-life of >6 h is surprisingly reduced to 1 h upon exposure of cells to As(III). These findings reveal a previously undescribed thiol reductase at the proteasome's regulatory particle
— id: 99210, year: 2009, vol: 284, page: 15233, stat: Journal Article,

Characterization of tafazzin splice variants from humans and fruit flies
Xu, Yang; Zhang, Shali; Malhotra, Ashim; Edelman-Novemsky, Irit; Ma, Jinping; Kruppa, Antonina; Cernicica, Carolina; Blais, Steven; Neubert, Thomas A; Ren, Mindong; Schlame, Michael
2009 Oct 16;284(42):29230-29239, Journal of biological chemistry
The tafazzin gene encodes a phospholipid-lysophospholipid transacylase involved in cardiolipin metabolism, but it is not known why it forms multiple transcripts as a result of alternative splicing. Here we studied the intracellular localization, enzymatic activity, and metabolic function of four isoforms of human tafazzin and three isoforms of Drosophila tafazzin upon expression in different mammalian and insect systems. When expressed in HeLa cells, all isoforms were localized in mitochondria except for the B-form of Drosophila tafazzin, which was associated with multiple intracellular membranes. Among the human isoforms, only full-length tafazzin (FL) and tafazzin lacking exon 5 (Delta5) had transacylase activity, and only these two isoforms were able to restore a normal cardiolipin pattern, normal respiratory activity of mitochondria, and male fertility in tafazzin-deficient flies. Both FL and Delta5 were associated with large protein complexes in 293T cell mitochondria, but treatment with alkali and proteinase K suggested that the Delta5 isoform was more integrated into the hydrophobic core of the membrane than the FL isoform. Although all Drosophila isoforms showed transacylase activity in vitro, only the A-form supported cardiolipin remodeling in flies. The data suggest that humans express two mitochondrial isoenzymes of tafazzin that have similar transacylase activities but different membrane topologies. Furthermore, the data show that the expression of human tafazzin in flies creates cardiolipin with a Drosophila pattern, suggesting that the characteristic fatty acid profile of cardiolipin is not determined by the substrate specificity of tafazzin
— id: 104345, year: 2009, vol: 284, page: 29230, stat: Journal Article,

Evaluation of the Variation in Sample Preparation for Comparative Proteomics Using Stable Isotope Labeling by Amino Acids in Cell Culture
Zhang, Guoan; Fenyo, David; Neubert, Thomas A
2009 Mar;8(3):1285-1292, Journal of proteome research
In comparative proteomic studies, it is important to know the variability associated with sample preparation. In this study, we report the strategy of using SILAC (stable isotope labeling by amino acids in cell culture) to evaluate the effect of the variation in sample preparation for quantitative proteomics. Variability can be measured when equal amounts of light and heavy SILAC samples undergo the same sample preparation procedures in parallel, and the two samples are mixed for relative protein quantitation by mass spectrometry. The high quantitative accuracy of SILAC allows for characterization of small variations. First, the reproducibility of immunoprecipitation (IP) and in-gel digestion was evaluated, and the impact of replicate number on quantitative accuracy was characterized. Second, we evaluated the overall variation in a comparative workflow involving three sequential sample preparation steps: IP, SDS-PAGE fractionation, and in-gel digestion. The evaluation of individual sample preparation steps was very valuable for experimental design: the optimal number of replicates for each step could be readily determined and the overall variation of the workflow could be predicted from the variation of the individual steps involved. By using informed experimental design, we demonstrated that the error associated with multiple steps of sample preparation in a comparative experiment can be limited to a reasonably low level
— id: 96814, year: 2009, vol: 8, page: 1285, stat: Journal Article,

Use of Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) for Phosphotyrosine Protein Identification and Quantitation
Zhang, Guoan; Neubert, Thomas A
2009 ;527:79-92, Methods in molecular biology
In recent years, stable isotope labeling by amino acids in cell culture (SILAC) has become increasingly popular as a quantitative proteomic method. In SILAC experiments, proteins are metabolically labeled by culturing cells in media containing normal and heavy isotope amino acids. This makes proteins from the light and heavy cells distinguishable by mass spectrometry (MS) after the cell lysates are mixed and the proteins separated and/or enriched. SILAC is a powerful tool for the study of intracellular signal transduction. In particular, it has been very popular and successful in quantitative analysis of phosphoty-rosine (pTyr) proteomes to characterize pTyr-dependent signaling pathways. In this chapter, we describe the SILAC procedure and use EphB signaling pathway as an example to illustrate the use of SILAC to investigate such pathways
— id: 96813, year: 2009, vol: 527, page: 79, stat: Journal Article,

Isoflurane inhibits cyclic adenosine monophosphate response element-binding protein phosphorylation and calmodulin translocation to the nucleus of SH-SY5Y cells
Zhang, Jin; Sutachan, Jhon-Jairo; Montoya-Gacharna, Jose; Xu, Chong-Feng; Xu, Fang; Neubert, Thomas A; Recio-Pinto, Esperanza; Blanck, Thomas J J
2009 Oct;109(4):1127-1134, Anesthesia & analgesia
BACKGROUND: Calmodulin (CaM) activation by Ca(2+), its translocation to the nucleus, and stimulation of phosphorylation of cyclic adenosine monophosphate response element-binding protein (CREB) (P-CREB) are necessary for new gene expression and have been linked to long-term potentiation, a process important in memory formation. Because isoflurane affects memory, we tested whether isoflurane interfered with the translocation of CaM to the neuronal cell nucleus and attenuated the formation P-CREB. METHODS: SH-SY5Y cells, a human neuroblastoma cell line, were cultured. Cells were depolarized with KCl and the phosphorylation of CREB examined by Western blotting, enzyme-linked immunosorbant assay, and immunocytochemistry. The translocation of CaM from the cytosol to the nucleus was also examined after depolarization. Cells were depolarized and lysed and fractionated by centrifugation to determine the amount of CaM translocated to the nucleus. CaM was localized by immunocytochemistry and quantitated by Western blotting and imaging. Before and during KCl depolarization, cells were exposed to isoflurane, isoflurane plus Bay K 8644, nitrendipine, and omega-conotoxin GVIa, respectively. RESULTS: P-CREB increased after KCl depolarization. The increase of P-CREB peaked at depolarization duration of 30 s. The increase in P-CREB formation was inhibited by nitrendipine, but not omega-conotoxin, and by isoflurane in a concentration-dependent fashion. Pretreatment with the L-type Ca(2+) channel agonist, Bay K 8644, attenuated the inhibition of P-CREB formation by isoflurane. CaM presence in the nucleus occurred after KCl depolarization. CaM translocation was inhibited by nitrendipine and attenuated by isoflurane. Bay K 8644 pretreatment decreased the isoflurane inhibition of CaM translocation to the nucleus. CONCLUSIONS: Our data demonstrate that isoflurane inhibits CaM translocation and P-CREB formation. This most likely occurs through isoflurane inhibition of Ca(2+)entry through L-type Ca(2+) channels
— id: 102500, year: 2009, vol: 109, page: 1127, stat: Journal Article,

A crystallographic snapshot of tyrosine trans-phosphorylation in action
Chen, Huaibin; Xu, Chong-Feng; Ma, Jinghong; Eliseenkova, Anna V; Li, Wanqing; Pollock, Pamela M; Pitteloud, Nelly; Miller, W Todd; Neubert, Thomas A; Mohammadi, Moosa
2008 Dec 16;105(50):19660-19665, Proceedings of the National Academy of Sciences of the United States of America
Tyrosine trans-phosphorylation is a key event in receptor tyrosine kinase signaling, yet, the structural basis for this process has eluded definition. Here, we present the crystal structure of the FGF receptor 2 kinases caught in the act of trans-phosphorylation of Y769, the major C-terminal phosphorylation site. The structure reveals that enzyme- and substrate-acting kinases engage each other through elaborate and specific interactions not only in the immediate vicinity of Y769 and the enzyme active site, but also in regions that are as much of 18 A away from D626, the catalytic base in the enzyme active site. These interactions lead to an unprecedented level of specificity and precision during the trans-phosphorylation on Y769. Time-resolved mass spectrometry analysis supports the observed mechanism of trans-phosphorylation. Our data provide a molecular framework for understanding the mechanism of action of Kallmann syndrome mutations and the order of trans-phosphorylation reactions in FGFRs. We propose that the salient mechanistic features of Y769 trans-phosphorylation are applicable to trans-phosphorylation of the equivalent major phosphorylation sites in many other RTKs
— id: 90927, year: 2008, vol: 105, page: 19660, stat: Journal Article,

ABRF-PRG05: de novo peptide sequence determination
Falick, Arnold M; Kowalak, Jeffrey A; Lane, William S; Phinney, Brett S; Turck, Christoph W; Weintraub, Susan T; West, Karen A; Neubert, Thomas A
2008 Sep;19(4):251-257, Journal of biomolecular techniques
A common request of proteomics core facilities is protein identification. However, in some instances primary sequence information for the protein in question is not present in public databases. In other cases, the amino acid sequence of a protein may differ in some way from the sequence predicted from the gene sequence in a database as a result of gene mutation, gene splicing, and/or multiple posttranslational modifications. Thus, it may be necessary to determine the sequence of one or more peptides de novo in order to identify and/or adequately characterize the protein of interest. The primary goal of this study was to give participating laboratories an opportunity to evaluate their proficiency in sequencing unknown peptides that are not included in any published database. Samples containing 3-6 pmol each of five synthetic peptides with amino acid sequences that were not present in public databases were sent to 106 laboratories. One nonstandard amino acid was present in one of the peptides. From a comparison of the results obtained by different strategies, participating laboratories will be able to gauge their own capabilities and establish realistic expectations for the approaches that can be used for this determination
— id: 96815, year: 2008, vol: 19, page: 251, stat: Journal Article,

Analysis of electroblotted proteins by mass spectrometry: protein identification after Western blotting
Luque-Garcia, Jose L; Zhou, Ge; Spellman, Daniel S; Sun, Tung-Tien; Neubert, Thomas A
2008 Feb;7(2):308-314, Molecular & cellular proteomics
We describe a new approach for the identification and characterization by mass spectrometry of proteins that have been electroblotted onto nitrocellulose. Using this method (Blotting and Removal of Nitrocellulose (BARN)), proteins can be analyzed either as intact proteins for molecular weight determination or as peptides generated by on-membrane proteolysis. Acetone is used to dissolve the nitrocellulose and to precipitate the adsorbed proteins/peptides, thus removing the nitrocellulose which can interfere with MS analysis. This method offers improved protein coverage, especially for membrane proteins, such as uroplakins, because the extraction step after in-gel digestion is avoided. Moreover, removal of nitrocellulose from the sample solution allows sample analysis by both MALDI- and (LC) ESI-based mass spectrometers. Finally, we demonstrate the utility of BARN for the direct identification of soluble and membrane proteins after Western blotting, obtaining comparable or better results than with in-gel digestion
— id: 76651, year: 2008, vol: 7, page: 308, stat: Journal Article,

Human Proteinpedia enables sharing of human protein data
Mathivanan, Suresh; Ahmed, Mukhtar; Ahn, Natalie G; Alexandre, Hainard; Amanchy, Ramars; Andrews, Philip C; Bader, Joel S; Balgley, Brian M; Bantscheff, Marcus; Bennett, Keiryn L; Bjorling, Erik; Blagoev, Blagoy; Bose, Ron; Brahmachari, Samir K; Burlingame, Alma S; Bustelo, Xose R; Cagney, Gerard; Cantin, Greg T; Cardasis, Helene L; Celis, Julio E; Chaerkady, Raghothama; Chu, Feixia; Cole, Philip A; Costello, Catherine E; Cotter, Robert J; Crockett, David; DeLany, James P; De Marzo, Angelo M; DeSouza, Leroi V; Deutsch, Eric W; Dransfield, Eric; Drewes, Gerard; Droit, Arnaud; Dunn, Michael J; Elenitoba-Johnson, Kojo; Ewing, Rob M; Van Eyk, Jennifer; Faca, Vitor; Falkner, Jayson; Fang, Xiangming; Fenselau, Catherine; Figeys, Daniel; Gagne, Pierre; Gelfi, Cecilia; Gevaert, Kris; Gimble, Jeffrey M; Gnad, Florian; Goel, Renu; Gromov, Pavel; Hanash, Samir M; Hancock, William S; Harsha, H C; Hart, Gerald; Hays, Faith; He, Fuchu; Hebbar, Prashantha; Helsens, Kenny; Hermeking, Heiko; Hide, Winston; Hjerno, Karin; Hochstrasser, Denis F; Hofmann, Oliver; Horn, David M; Hruban, Ralph H; Ibarrola, Nieves; James, Peter; Jensen, Ole N; Jensen, Pia Honnerup; Jung, Peter; Kandasamy, Kumaran; Kheterpal, Indu; Kikuno, Reiko F; Korf, Ulrike; Korner, Roman; Kuster, Bernhard; Kwon, Min-Seok; Lee, Hyoung-Joo; Lee, Young-Jin; Lefevre, Michael; Lehvaslaiho, Minna; Lescuyer, Pierre; Levander, Fredrik; Lim, Megan S; Lobke, Christian; Loo, Joseph A; Mann, Matthias; Martens, Lennart; Martinez-Heredia, Juan; McComb, Mark; McRedmond, James; Mehrle, Alexander; Menon, Rajasree; Miller, Christine A; Mischak, Harald; Mohan, S Sujatha; Mohmood, Riaz; Molina, Henrik; Moran, Michael F; Morgan, James D; Moritz, Robert; Morzel, Martine; Muddiman, David C; Nalli, Anuradha; Navarro, J Daniel; Neubert, Thomas A; Ohara, Osamu; Oliva, Rafael; Omenn, Gilbert S; Oyama, Masaaki; Paik, Young-Ki; Pennington, Kyla; Pepperkok, Rainer; Periaswamy, Balamurugan; Petricoin, Emanuel F; Poirier, Guy G; Prasad, T S Keshava; Purvine, Samuel O; Rahiman, B Abdul; Ramachandran, Prasanna; Ramachandra, Y L; Rice, Robert H; Rick, Jens; Ronnholm, Ragna H; Salonen, Johanna; Sanchez, Jean-Charles; Sayd, Thierry; Seshi, Beerelli; Shankari, Kripa; Sheng, Shi Jun; Shetty, Vivekananda; Shivakumar, K; Simpson, Richard J; Sirdeshmukh, Ravi; Siu, K W Michael; Smith, Jeffrey C; Smith, Richard D; States, David J; Sugano, Sumio; Sullivan, Matthew; Superti-Furga, Giulio; Takatalo, Maarit; Thongboonkerd, Visith; Trinidad, Jonathan C; Uhlen, Mathias; Vandekerckhove, Joel; Vasilescu, Julian; Veenstra, Timothy D; Vidal-Taboada, Jose-Manuel; Vihinen, Mauno; Wait, Robin; Wang, Xiaoyue; Wiemann, Stefan; Wu, Billy; Xu, Tao; Yates, John R; Zhong, Jun; Zhou, Ming; Zhu, Yunping; Zurbig, Petra; Pandey, Akhilesh
2008 Feb;26(2):164-167, Nature biotechnology
— id: 76647, year: 2008, vol: 26, page: 164, stat: Journal Article,

Calsyntenins Are Secretory Granule Proteins in Anterior Pituitary Gland and Pancreatic Islet {alpha} Cells
Rindler, Michael J; Xu, Chong-Feng; Gumper, Iwona; Cen, Chuan; Sonderegger, Peter; Neubert, Thomas A
2008 Apr;56(4):381-388, Journal of histochemistry & cytochemistry
Calsyntenins are members of the cadherin superfamily of cell adhesion molecules. They are present in postsynaptic membranes of excitatory neurons and in vesicles in transit to neuronal growth cones. In the current study, calsyntenin-1 (CST-1) and calsyntenin-3 (CST-3) were identified by mass spectrometric analysis (LC-MS/MS) of integral membrane proteins from highly enriched secretory granule preparations from bovine anterior pituitary gland. Immunofluorescence microscopy on thin frozen sections of rat pituitary revealed that CST-1 was present only in gonadotropes where it colocalized with follicle-stimulating hormone in secretory granules. In contrast, CST-3 was present not only in gonadotrope secretory granules but also in those of somatotropes and thyrotropes. Neither protein was detected in mammatropes. In addition, CST-1 was also localized to the glucagon-containing secretory granules of alpha cells in the pancreatic islets of Langerhans. Results indicate that calsyntenins function outside the nervous system and potentially are modulators of endocrine function
— id: 76650, year: 2008, vol: 56, page: 381, stat: Journal Article,

Stable isotopic labeling of amino acids in cultured primary neurons: Application to BDNF-dependent phosphotyrosine-associated signaling
Spellman, Daniel S; Deinhardt, Katrin; Darie, Costel C; Chao, Moses V; Neubert, Thomas A
2008 Jun;7(6):1067-1076, Molecular & cellular proteomics
Cultured primary neurons are a well established model for the study of neuronal function in vitro. Here we have demonstrated that Stable Isotope Labeling by Amino Acids in Cell culture (SILAC) can be applied to a differentiated, non-dividing cell type such as primary neurons, and we have applied this technique to assess changes in the neuronal phosphotyrosine proteome in response to stimulation by BDNF (brain derived neurotrophic factor), an important molecule for the development and regulation of neuronal connections. We found that 13 proteins had SILAC ratios above 1.50 or below 0.67 in phosphotyrosine immunoprecipitates (pY IPs) comparing BDNF-treated and control samples, and an additional 18 proteins had ratios above 1.25 or below 0.80. These proteins include TrkB, the receptor tyrosine kinase (RTK) for BDNF, and others such as Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and STAM (signal-transducing adaptor molecule), which are proteins known to regulate intracellular trafficking of RTKs. These results demonstrate that the combination of primary neuronal cell culture and SILAC can be a powerful tool for the study of the proteomes of neuronal molecular and cellular dynamics
— id: 76648, year: 2008, vol: 7, page: 1067, stat: Journal Article,

Guidelines for reporting the use of mass spectrometry in proteomics
Taylor, Chris F; Binz, Pierre-Alain; Aebersold, Ruedi; Affolter, Michel; Barkovich, Robert; Deutsch, Eric W; Horn, David M; Huhmer, Andreas; Kussmann, Martin; Lilley, Kathryn; Macht, Marcus; Mann, Matthias; Muller, Dieter; Neubert, Thomas A; Nickson, Janice; Patterson, Scott D; Raso, Roberto; Resing, Kathryn; Seymour, Sean L; Tsugita, Akira; Xenarios, Ioannis; Zeng, Rong; Julian, Randall K Jr
2008 Aug;26(8):860-861, Nature biotechnology
— id: 96817, year: 2008, vol: 26, page: 860, stat: Journal Article,

Phosphorylation of liver X receptor alpha selectively regulates target gene expression in macrophages
Torra, Ines Pineda; Ismaili, Naima; Feig, Jonathan E; Xu, Chong-Feng; Cavasotto, Claudio; Pancratov, Raluca; Rogatsky, Inez; Neubert, Thomas A; Fisher, Edward A; Garabedian, Michael J
2008 Apr;28(8):2626-2636, Molecular & cellular biology
Dysregulation of liver X receptor alpha (LXRalpha) activity has been linked to cardiovascular and metabolic diseases. Here, we show that LXRalpha target gene selectivity is achieved by modulation of LXRalpha phosphorylation. Under basal conditions, LXRalpha is phosphorylated at S198; phosphorylation is enhanced by LXR ligands and reduced both by casein kinase 2 (CK2) inhibitors and by activation of its heterodimeric partner RXR with 9-cis-retinoic acid (9cRA). Expression of some (AIM and LPL), but not other (ABCA1 or SREBPc1) established LXR target genes is increased in RAW 264.7 cells expressing the LXRalpha S198A phosphorylation-deficient mutant compared to those with WT receptors. Surprisingly, a gene normally not expressed in macrophages, the chemokine CCL24, is activated specifically in cells expressing LXRalpha S198A. Furthermore, inhibition of S198 phosphorylation by 9cRA or by a CK2 inhibitor similarly promotes CCL24 expression, thereby phenocopying the S198A mutation. Thus, our findings reveal a previously unrecognized role for phosphorylation in restricting the repertoire of LXRalpha-responsive genes
— id: 76646, year: 2008, vol: 28, page: 2626, stat: Journal Article,

Histoplasma capsulatum proteome response to decreased iron availability
Winters, Michael S; Spellman, Daniel S; Chan, Qilin; Gomez, Francisco J; Hernandez, Margarita; Catron, Brittany; Smulian, Alan G; Neubert, Thomas A; Deepe, George S Jr
2008 ;6:36-36, Proteome science
ABSTRACT: BACKGROUND: A fundamental pathogenic feature of the fungus Histoplasma capsulatum is its ability to evade innate and adaptive immune defenses. Once ingested by macrophages the organism is faced with several hostile environmental conditions including iron limitation. H. capsulatum can establish a persistent state within the macrophage. A gap in knowledge exists because the identities and number of proteins regulated by the organism under host conditions has yet to be defined. Lack of such knowledge is an important problem because until these proteins are identified it is unlikely that they can be targeted as new and innovative treatment for histoplasmosis. RESULTS: To investigate the proteomic response by H. capsulatum to decreasing iron availability we have created H. capsulatum protein/genomic databases compatible with current mass spectrometric (MS) search engines. Databases were assembled from the H. capsulatum G217B strain genome using gene prediction programs and expressed sequence tag (EST) libraries. Searching these databases with MS data generated from two dimensional (2D) in-gel digestions of proteins resulted in over 50% more proteins identified compared to searching the publicly available fungal databases alone. Using 2D gel electrophoresis combined with statistical analysis we discovered 42 H. capsulatum proteins whose abundance was significantly modulated when iron concentrations were lowered. Altered proteins were identified by mass spectrometry and database searching to be involved in glycolysis, the tricarboxylic acid cycle, lysine metabolism, protein synthesis, and one protein sequence whose function was unknown. CONCLUSION: We have created a bioinformatics platform for H. capsulatum and demonstrated the utility of a proteomic approach by identifying a shift in metabolism the organism utilizes to cope with the hostile conditions provided by the host. We have shown that enzyme transcripts regulated by other fungal pathogens in response to lowering iron availability are also regulated in H. capsulatum at the protein level. We also identified H. capsulatum proteins sensitive to iron level reductions which have yet to be connected to iron availability in other pathogens. These data also indicate the complexity of the response by H. capsulatum to nutritional deprivation. Finally, we demonstrate the importance of a strain specific gene/protein database for H. capsulatum proteomic analysis
— id: 96816, year: 2008, vol: 6, page: 36, stat: Journal Article,

Structural and biochemical characterization of the KRLB region in insulin receptor substrate-2
Wu, Jinhua; Tseng, Yolanda D; Xu, Chong-Feng; Neubert, Thomas A; White, Morris F; Hubbard, Stevan R
2008 Mar;15(3):251-258, Nature structural & molecular biology
Insulin receptor substrates 1 and 2 (IRS1 and -2) are crucial adaptor proteins in mediating the metabolic and mitogenic effects of insulin and insulin-like growth factor 1. These proteins consist of a pleckstrin homology domain, a phosphotyrosine binding domain and a C-terminal region containing numerous sites of tyrosine, serine and threonine phosphorylation. Previous yeast two-hybrid studies identified a region unique to IRS2, termed the kinase regulatory-loop binding (KRLB) region, which interacts with the tyrosine kinase domain of the insulin receptor. Here we present the crystal structure of the insulin receptor kinase in complex with a 15-residue peptide from the KRLB region. In the structure, this segment of IRS2 is bound in the kinase active site with Tyr628 positioned for phosphorylation. Although Tyr628 was phosphorylated by the insulin receptor, its catalytic turnover was poor, resulting in kinase inhibition. Our studies indicate that the KRLB region functions to limit tyrosine phosphorylation of IRS2
— id: 76468, year: 2008, vol: 15, page: 251, stat: Journal Article,

Proteasomal adaptation to environmental stress links resistance to proteotoxicity with longevity in Caenorhabditis elegans
Yun, Chi; Stanhill, Ariel; Yang, Yun; Zhang, Yuhong; Haynes, Cole M; Xu, Chong-Feng; Neubert, Thomas A; Mor, Adam; Philips, Mark R; Ron, David
2008 May 13;105(19):7094-7099, Proceedings of the National Academy of Sciences of the United States of America
The burden of protein misfolding is believed to contribute to aging. However, the links between adaptations to conditions associated with protein misfolding and resistance to the time-dependent attrition of cellular function remain poorly understood. We report that worms lacking aip-1, a homologue of mammalian AIRAP (arsenic-inducible proteasomal 19S regulatory particle-associated protein), are not only impaired in their ability to resist exposure to arsenite but also exhibit shortened lifespan and hypersensitivity to misfolding-prone proteins under normal laboratory conditions. Mammals have a second, constitutively expressed AIRAP-like gene (AIRAPL) that also encodes a proteasome-interacting protein, which shares with AIRAP the property of enhancing peptide accessibility to the proteasome's active site. Genetic rescue experiments suggest that features common to the constitutively expressed worm AIP-1 and mammalian AIRAPL (but missing in the smaller, arsenite-inducible AIRAP) are important to lifespan extension. In worms, a single AIRAP-related protein links proteasomal adaptation to environmental stress with resistance to both proteotoxic insults and maintenance of animal life span under normal conditions
— id: 94504, year: 2008, vol: 105, page: 7094, stat: Journal Article,

Screening for EphB signaling effectors using SILAC with a linear ion trap-orbitrap mass spectrometer
Zhang, Guoan; Fenyo, David; Neubert, Thomas A
2008 Nov;7(11):4715-4726, Journal of proteome research
Erythropoietin-producing hepatocellular carcinoma (Eph) receptors play important roles in development, neural plasticity, and cancer. We used an Orbitrap mass spectrometer and stable isotope labeling by amino acids in cell culture (SILAC) to identify and quantify 204 proteins with significantly changed abundance in antiphosphotyrosine immunoprecipitates after ephrinB1-Fc stimulation. More than half of all known effectors downstream of EphB receptors were identified in this study, as well as numerous novel candidates for EphB signaling
— id: 91446, year: 2008, vol: 7, page: 4715, stat: Journal Article,

Use of DNA ladders for reproducible protein fractionation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) for quantitative proteomics
Zhang, Guoan; Fenyo, David; Neubert, Thomas A
2008 Feb;7(2):678-686, Journal of proteome research
In proteomics, one-dimensional (1D) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is widely used for protein fractionation prior to mass spectrometric analysis to enhance the dynamic range of analysis and to improve the identification of low-abundance proteins. Such protein prefractionation works well for quantitation strategies if the proteins are labeled prior to separation. However, because of the poor reproducibility of cutting gel slices, especially when small amounts of samples are analyzed, its application in label-free and peptide-labeling quantitative proteomics methods has been greatly limited. To overcome this limitation, we developed a new strategy in which a DNA ladder is mixed with the protein sample before PAGE separation. After PAGE separation, the DNA ladder is stained to allow for easy, precise, and reproducible gel cutting. To this end, a novel visible DNA-staining method was developed. This staining method is fast, sensitive, and compatible with mass spectrometry. To evaluate the reproducibility of DNA-ladder-assisted gel cutting for quantitative protein fractionation, we used stable isotope labeling with amino acids in cell culture (SILAC). Our results show that the quantitative error associated with fractionation can be minimized using the DNA-assisted fractionation and multiple replicates of gel cutting. In conclusion, 1D PAGE fractionation in combination with DNA ladders can be used for label-free comparative proteomics without compromising quantitation
— id: 76649, year: 2008, vol: 7, page: 678, stat: Journal Article,

ABRF-PRG04: differentiation of protein isoforms
Arnott, David; Gawinowicz, Mary Ann; Kowalak, Jeffrey A; Lane, William S; Speicher, Kaye D; Turck, Christoph W; West, Karen A; Neubert, Thomas A
2007 Apr;18(2):124-134, Journal of biomolecular techniques
Accurate protein identification sometimes requires careful discrimination between closely related protein isoforms that may differ by as little as a single amino acid substitution or post-translational modification. The ABRF Proteomics Research Group sent a mixture of three picomoles each of three closely related proteins to laboratories who requested it in the form of intact proteins, and participating laboratories were asked to identify the proteins and report their results. The primary goal of the ABRF-PRG04 Study was to give participating laboratories a chance to evaluate their capabilities and practices with regards to sample fractionation (1D- or 2D-PAGE, HPLC, or none), protein digestion methods (in-solution, in-gel, enzyme choice), and approaches to protein identification (instrumentation, use of software, and/or manual techniques to facilitate interpretation), as well as determination of amino acid or post-translational modifications. Of the 42 laboratories that responded, 8 (19%) correctly identified all three isoforms and N-terminal acetylation of each, 16 (38%) labs correctly identified two isoforms, 9 (21%) correctly identified two isoforms but also made at least one incorrect identification, and 9 (21%) made no correct protein identifications. All but one lab used mass spectrometry, and data submitted enabled a comparison of strategies and methods used.
— id: 72970, year: 2007, vol: 18, page: 124, stat: Journal Article,

A molecular brake in the kinase hinge region regulates the activity of receptor tyrosine kinases
Chen, Huaibin; Ma, Jinghong; Li, Wanqing; Eliseenkova, Anna V; Xu, Chongfeng; Neubert, Thomas A; Miller, W Todd; Mohammadi, Moosa
2007 Sep 7;27(5):717-730, Molecular cell
Activating mutations in the tyrosine kinase domain of receptor tyrosine kinases (RTKs) cause cancer and skeletal disorders. Comparison of the crystal structures of unphosphorylated and phosphorylated wild-type FGFR2 kinase domains with those of seven unphosphorylated pathogenic mutants reveals an autoinhibitory 'molecular brake' mediated by a triad of residues in the kinase hinge region of all FGFRs. Structural analysis shows that many other RTKs, including PDGFRs, VEGFRs, KIT, CSF1R, FLT3, TEK, and TIE, are also subject to regulation by this brake. Pathogenic mutations activate FGFRs and other RTKs by disengaging the brake either directly or indirectly
— id: 73939, year: 2007, vol: 27, page: 717, stat: Journal Article,

Molecular insights into the klotho-dependent, endocrine mode of action of fibroblast growth factor 19 subfamily members
Goetz, Regina; Beenken, Andrew; Ibrahimi, Omar A; Kalinina, Juliya; Olsen, Shaun K; Eliseenkova, Anna V; Xu, ChongFeng; Neubert, Thomas A; Zhang, Fuming; Linhardt, Robert J; Yu, Xijie; White, Kenneth E; Inagaki, Takeshi; Kliewer, Steven A; Yamamoto, Masaya; Kurosu, Hiroshi; Ogawa, Yasushi; Kuro-o, Makoto; Lanske, Beate; Razzaque, Mohammed S; Mohammadi, Moosa
2007 May;27(9):3417-3428, Molecular & cellular biology
Unique among fibroblast growth factors (FGFs), FGF19, -21, and -23 act in an endocrine fashion to regulate energy, bile acid, glucose, lipid, phosphate, and vitamin D homeostasis. These FGFs require the presence of Klotho/betaKlotho in their target tissues. Here, we present the crystal structures of FGF19 alone and FGF23 in complex with sucrose octasulfate, a disaccharide chemically related to heparin. The conformation of the heparin-binding region between beta strands 10 and 12 in FGF19 and FGF23 diverges completely from the common conformation adopted by paracrine-acting FGFs. A cleft between this region and the beta1-beta2 loop, the other heparin-binding region, precludes direct interaction between heparin/heparan sulfate and backbone atoms of FGF19/23. This reduces the heparin-binding affinity of these ligands and confers endocrine function. Klotho/betaKlotho have evolved as a compensatory mechanism for the poor ability of heparin/heparan sulfate to promote binding of FGF19, -21, and -23 to their cognate receptors
— id: 71392, year: 2007, vol: 27, page: 3417, stat: Journal Article,

Sample preparation for serum/plasma profiling and biomarker identification by mass spectrometry
Luque-Garcia, Jose L; Neubert, Thomas A
2007 Jun 15;1153(1-2):259-276, Journal of chromatography
In this article, we present an overview of the different strategies for sample preparation for identification by mass spectrometry (MS) of biomarkers from serum and/or plasma. We consider the effects of the variables involved in sample collection, handling and storage, and describe different approaches for removal of high abundance proteins and serum/plasma fractionation. We review the advantages and disadvantages of such techniques as centrifugal ultrafiltration, different formats for solid phase extraction, organic solvent extraction, gel and capillary electrophoresis, and liquid chromatography. We also discuss a variety of current proteomic methods and their main applications for biomarker-related studies
— id: 71394, year: 2007, vol: 1153, page: 259, stat: Journal Article,

Using SILAC to Study Cell Signaling in Neurons
Neubert, Thomas A
[S.l.] : NIH, 2007,
The formation and refinement of connections between neurons in the developing brain, and modulation of synaptic strength in the adult brain often rely on the stimulation of receptor tyrosine kinases by their ligands. Much can be learned about how this signaling works by using stable isotope labeling quantitative proteomic methods such as Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and targeted protein isolation strategies. These sample preparation methods are then combined with liquid chromatography-Q-TOF or LTQ-Orbitrap tandem mass spectrometry to identify and compare the relative amounts of proteins in signal transduction complexes from stimulated and nonstimulated cells. I will describe the use of SILAC in our lab to study ephrin signaling in NG108 cell cultures and BDNF signaling in primary cortical neurons. I will also describe SILAC experiments to characterize activity-dependent changes in postsynaptic density composition in primary neuronal cell culture
— id: 1432, year: 2007, vol: , page: , stat: ,

Proteomic analysis of exfoliation deposits
Ovodenko, Boris; Rostagno, Agueda; Neubert, Thomas A; Shetty, Vivekananda; Thomas, Stefani; Yang, Austin; Liebmann, Jeffrey; Ghiso, Jorge; Ritch, Robert
2007 Apr;48(4):1447-1457, Investigative ophthalmology & visual science. IOVS
PURPOSE: To increase knowledge of the biochemical composition of lenticular exfoliation material (XFM) by using proteomic approaches. METHODS: Anterior lens capsules from patients with and without exfoliation syndrome (XFS) were homogenized in formic acid and subjected to cyanogen bromide (CNBr) cleavage, and the pattern of chemically generated fragments was compared by SDS-PAGE after silver staining. Unique XFS bands not present in control cases were excised, digested with TPCK-trypsin, and the resultant peptides sequenced with quadrupole time-of-flight mass spectrometry (MS). In parallel experiments, CNBr-fragmented XFM was separately digested in solution with trypsin and elastase, and the resultant peptide mixture was analyzed by liquid chromatography coupled to tandem MS followed by identification through homology searches at nonredundant protein databases. Immunolocalization of the MS-identified components were performed in XFS versus control samples by using conventional immunohistochemical methods and light microscopy. RESULTS: In addition to fibrillin-1, fibronectin, vitronectin, laminin, and amyloid P-component, which are well-known extracellular matrix and basement membrane components of XFM, the proteomic approaches identified the multifunctional protein clusterin and tissue inhibitor of metalloprotease (TIMP)-3 as well as novel molecules, among them fibulin-2, desmocollin-2, the glycosaminoglycans syndecan-3, and versican, membrane metalloproteases of the ADAM family (a disintegrin and metalloprotease), and the initiation component of the classic complement activation pathway C1q. In all cases, classic immunohistochemistry confirmed their location in XFM. CONCLUSIONS: A novel solubilization strategy combined with sensitive proteomic analysis emphasizes the complexity of the XFS deposits and opens new avenues to study the molecular mechanisms involved in the pathogenesis and progression of XFS
— id: 71391, year: 2007, vol: 48, page: 1447, stat: Journal Article,

Proteomic Analysis of Pancreatic Zymogen Granules: Identification of New Granule Proteins
Rindler, Michael J; Xu, Chong-Feng; Gumper, Iwona; Smith, Nora N; Neubert, Thomas A
2007 Aug;6(8):2978-2992, Journal of proteome research
The composition of zymogen granules from rat pancreas was determined by LC-MS/MS. Enriched intragranular content, peripheral membrane, and integral membrane protein fractions were analyzed after one-dimensional SDS-PAGE and tryptic digestion of gel slices. A total of 371 proteins was identified with high confidence, including 84 previously identified granule proteins. The 287 remaining proteins included 37 GTP-binding proteins and effectors, 8 tetraspan membrane proteins, and 22 channels and transporters. Seven proteins, pantophysin, cyclic nucleotide phosphodiesterase, carboxypeptidase D, ecto-nucleotide phosphodiesterase 3, aminopeptidase N, ral, and the potassium channel TWIK-2, were confirmed by immunofluorescence microscopy or by immunoblotting to be new zymogen granule membrane proteins. Keywords: proteomics * mass spectrometry * LC-MS/MS * pancreas * zymogen granules * acinar cells.
— id: 72969, year: 2007, vol: 6, page: 2978, stat: Journal Article,

Characterization by tandem mass spectrometry of stable cysteine sulfenic acid in a cysteine switch peptide of matrix metalloproteinases
Shetty, Vivekananda; Spellman, Daniel S; Neubert, Thomas A
2007 Aug;18(8):1544-1551, Journal of the American Society for Mass Spectrometry
Cysteine sulfenic acid (Cys-SOH) is an elusive intermediate in reactive oxygen species-induced oxidation reactions of many proteins such as peroxiredoxins and tyrosine phosphatases. Cys-SOH is proposed to play a vital role in catalytic and signaling functions. The formation of cysteine sulfinic acid (Cys-SO(2)H) and cysteine sulfonic acid (Cys-SO(3)H) has been implicated in the activation of matrix metalloproteinase-7 (MMP-7) and oxidation of thiol to cysteine sulfinic acid has been associated with the autolytic cleavage of MMP-7. We have examined the formation of cysteine sulfenic acid in a synthetic peptide PRCGVPDVA, which is a cysteine switch domain of MMP-7 and other matrix metalloproteases. We have prepared the cysteine sulfenic acid containing peptide, PRC(SOH)GVPDVA, by reaction with hydroxyl radicals generated by the Fenton reaction (Fe(+2)/H(2)O(2)). We characterized this modified peptide by tandem mass spectrometry and accurate mass measurement experiments. In addition, we used 7-chloro-4-nitrobenzo-2-oxa-1,3-diazol (NBD-Cl) reagent to form an adduct with PRC(SOH)GVPDVA to provide additional evidence for the viability of PRC(SOH)GVPDVA in solution. We also characterized an intramolecular cysteine sulfinamide cross-link product PRC[S(O)N]GVPDVA based on tandem mass spectrometry and accurate mass measurement experiments. These results contribute to the understanding of a proteolytic cleavage mechanism that is traditionally associated with MMP activation
— id: 73855, year: 2007, vol: 18, page: 1544, stat: Journal Article,

The minimum information about a proteomics experiment (MIAPE)
Taylor, Chris F; Paton, Norman W; Lilley, Kathryn S; Binz, Pierre-Alain; Julian, Randall K Jr; Jones, Andrew R; Zhu, Weimin; Apweiler, Rolf; Aebersold, Ruedi; Deutsch, Eric W; Dunn, Michael J; Heck, Albert J R; Leitner, Alexander; Macht, Marcus; Mann, Matthias; Martens, Lennart; Neubert, Thomas A; Patterson, Scott D; Ping, Peipei; Seymour, Sean L; Souda, Puneet; Tsugita, Akira; Vandekerckhove, Joel; Vondriska, Thomas M; Whitelegge, Julian P; Wilkins, Marc R; Xenarios, Ioannnis; Yates, John R 3rd; Hermjakob, Henning
2007 Aug;25(8):887-893, Nature biotechnology
Both the generation and the analysis of proteomics data are now widespread, and high-throughput approaches are commonplace. Protocols continue to increase in complexity as methods and technologies evolve and diversify. To encourage the standardized collection, integration, storage and dissemination of proteomics data, the Human Proteome Organization's Proteomics Standards Initiative develops guidance modules for reporting the use of techniques such as gel electrophoresis and mass spectrometry. This paper describes the processes and principles underpinning the development of these modules; discusses the ramifications for various interest groups such as experimentalists, funders, publishers and the private sector; addresses the issue of overlap with other reporting guidelines; and highlights the criticality of appropriate tools and resources in enabling 'MIAPE-compliant' reporting
— id: 73905, year: 2007, vol: 25, page: 887, stat: Journal Article,

Selective enrichment and fractionation of phosphopeptides from peptide mixtures by isoelectric focusing after methyl esterification
Xu, Chong-Feng; Wang, Huaibin; Li, Daming; Kong, Xiang-Peng; Neubert, Thomas A
2007 Mar 1;79(5):2007-2014, Analytical chemistry
We have developed a new strategy to enrich and fractionate phosphopeptides from peptide mixtures based on the difference in their isoelectric points (pIs) after methyl esterification. After isoelectric focusing (IEF) of a methylated tryptic digest of a mixture of alpha-S-casein and beta-casein, phosphopeptides were selectively enriched at acidic and neutral pHs while nonphosphopeptides left the focusing gel because their pIs are higher than the upper limit of the immobilized pH gradient. We wrote a web-based program, pIMethylation, to predict the pIs for peptides with and without methyl esterification. Theoretical calculations using pIMethylation indicated that methylated phosphopeptides and non-phosphopeptides can be grouped on the basis of the number of phosphate groups and basic residues in each peptide. Our IEF results were consistent with theoretical pIs of methylated peptides calculated by pIMethylation. We also showed that 2,6-dihydroxy-acetophenone is superior to 2,5-dihydroxybenzoic acid as a matrix for MALDI Q-TOF MS of methylated phosphopeptides in both positive and negative ion modes
— id: 71393, year: 2007, vol: 79, page: 2007, stat: Journal Article,

Use of nitrocellulose membranes for protein characterization by matrix-assisted laser desorption/ionization mass spectrometry
Luque-Garcia, Jose L; Zhou, Ge; Sun, Tung-Tien; Neubert, Thomas A
2006 Jul 15;78(14):5102-5108, Analytical chemistry
We present an improved method for MALDI-MS analysis of proteins that have been electroblotted onto a nitrocellulose (NC) membrane. With this approach, electroblotted proteins can be analyzed directly for intact molecular weight determination or after on-membrane digestion by dissolution of the nitrocellulose in MALDI matrix solution containing 70% acetonitrile and 30% methanol. This solution helps maintain solubility of proteins and peptides while dissolving the NC membrane, which is dissolved by 100% acetone in other protocols. On-membrane tryptic digestion using this method requires half the time of in-gel digestion and results in fewer missed cleavages and better protein coverage. For the membrane proteins studied, bovine uroplakins II and III, the protein coverage was almost twice that provided by conventional in-gel digestion, and the transmembrane domains of both uroplakins were detected only after on-membrane digestion. We also demonstrated the compatibility with MALDI-MS of a new dye, MemCode, which is specifically designed for staining NC membrane-immobilized proteins and is faster and more sensitive than Ponceau-S. Our improved on-membrane digestion protocol greatly improves the study of soluble and, particularly strikingly, integral membrane proteins by mass spectrometry
— id: 71579, year: 2006, vol: 78, page: 5102, stat: Journal Article,

Automated comparative proteomics based on multiplex tandem mass spectrometry and stable isotope labeling
Zhang, Guoan; Neubert, Thomas A
2006 Feb;5(2):401-411, Molecular & cellular proteomics
Comparative proteomic approaches using isotopic labeling and mass spectrometry (MS) have become increasingly popular. Conventionally, quantification is based on MS or extracted ion chromatogram (XIC) signals of differentially labeled peptides. However, in these MS-based experiments, the accuracy and dynamic range of quantification are limited by the high noise levels of MS/XIC data. Here we report a quantitative strategy based on multiplex (derived from multiple precursor ions) MS/MS data. One set of proteins was metabolically labeled with 13C6 lysine and 15N4 arginine, the other set unlabeled. For peptide analysis after tryptic digestion of the labeled proteins, a wide precursor window was used to include both the light and heavy versions of each peptide for fragmentation. The multiplex MS/MS data were used for both protein identification and quantification. The use of the wide precursor window increased sensitivity and the y ion pairs in the multiplex MS/MS spectra from peptides containing labeled and unlabeled lysine or arginine offered more information for, and thus the potential for improving, protein identification. Protein ratios were obtained by comparing intensities of y ions derived from the light and heavy peptides. Our results indicated that this method offers several advantages over the conventional XIC-based approach, including increased sensitivity for protein identification and more accurate quantification with more than a ten-fold increase in dynamic range. In addition, the quantification calculation process was fast, fully automated and independent of instrument and data type. This method was further validated by quantitative analysis of signaling proteins in the EphB2 pathway in NG-108 cells
— id: 61371, year: 2006, vol: 5, page: 401, stat: Journal Article,

Use of detergents to increase selectivity of immunoprecipitation of tyrosine phosphorylated peptides prior to identification by MALDI quadrupole-TOF MS
Zhang, Guoan; Neubert, Thomas A
2006 Jan;6(2):571-578, Proteomics
Identification of tyrosine phosphorylation by MS is challenging due to its low abundance in biological samples. Therefore, specific enrichment of tyrosine phosphorylated peptides prior to their analysis is highly desirable. The application of immunopurification of phosphotyrosine (pY) peptides using pY antibodies has been greatly limited by poor selectivity. In the present study, we have shown that the selectivity of pY peptide immunopurification can be dramatically improved by adding detergents to immunoprecipitation buffers. Optimum selectivity and sensitivity were achieved using an immunoprecipitation buffer containing n-octyl glucoside with a concentration above its critical micelle concentration (0.7%). The optimized method was used to identify in vivo tyrosine phosphorylation on proteins isolated from cell extract by anti-pY protein immunoprecipitation. After immunopurification, non-pY-containing peptides from protein digests were readily removed and pY peptides became the dominant peaks in MALDI quadrupole-TOF mass spectra. In addition, the signal intensities from pY-containing peptides were enhanced significantly after enrichment, allowing characterization of tyrosine phosphorylation sites with greater sensitivity
— id: 76653, year: 2006, vol: 6, page: 571, stat: Journal Article,

Quantitative phosphotyrosine proteomics of EphB2 signaling by stable isotope labeling with amino acids in cell culture (SILAC)
Zhang, Guoan; Spellman, Daniel S; Skolnik, Edward Y; Neubert, Thomas A
2006 Mar;5(3):581-588, Journal of proteome research
Eph-related receptor tyrosine kinases (RTK) have been implicated in several biological functions including synaptic plasticity, axon guidance, and morphogenesis, yet the details of the signal transduction pathways that produce these specific biological functions after ligand-receptor interaction remain unclear. We used Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) in combination with LC-MS/MS to characterize cellular signaling following stimulation by ephrinB1-Fc of NG-108 cells that overexpress EphB2 receptors. Because tyrosine phosphorylation functions as a key regulatory event in RTK signaling, we used anti-phosphotyrosine immunoprecipitation (pY IP) of cell lysates to isolate potential participants in the EphB2 pathway. Our SILAC experiments identified 127 unique proteins, 40 of which demonstrated increased abundance in pY IPs from ephrinB1-Fc stimulated cells as compared with unstimulated cells. Six proteins demonstrated decreased abundance, and 81 did not change significantly in relative abundance. Western blotting analysis of five proteins after pY IP verified their SILAC results. On the basis of previously published work and use of PathwayAssist software, we proposed an interaction network downstream of EphB2 for the proteins with changed ratios
— id: 76652, year: 2006, vol: 5, page: 581, stat: Journal Article,

Familial Danish dementia: co-existence of Danish and Alzheimer amyloid subunits (ADan AND A{beta}) in the absence of compact plaques
Tomidokoro, Yasushi; Lashley, Tammaryn; Rostagno, Agueda; Neubert, Thomas A; Bojsen-Moller, Marie; Braendgaard, Hans; Plant, Gordon; Holton, Janice; Frangione, Blas; Revesz, Tamas; Ghiso, Jorge
2005 Nov 4;280(44):36883-36894, Journal of biological chemistry
Familial Danish dementia is an early onset autosomal dominant neurodegenerative disorder linked to a genetic defect in the BRI2 gene and clinically characterized by dementia and ataxia. Cerebral amyloid and preamyloid deposits of two unrelated molecules (Danish amyloid (ADan) and beta-amyloid (Abeta)), the absence of compact plaques, and neurofibrillary degeneration indistinguishable from that observed in Alzheimer disease (AD) are the main neuropathological features of the disease. Biochemical analysis of extracted amyloid and preamyloid species indicates that as the solubility of the deposits decreases, the heterogeneity and complexity of the extracted peptides exponentially increase. Nonfibrillar deposits were mainly composed of intact ADan-(1-34) and its N-terminally modified (pyroglutamate) counterpart together with Abeta-(1-42) and Abeta-(4-42) in approximately 1:1 mixture. The post-translational modification, glutamate to pyroglutamate, was not present in soluble circulating ADan. In the amyloid fractions, ADan was heavily oligomerized and highly heterogeneous at the N and C terminus, and, when intact, its N terminus was post-translationally modified (pyroglutamate), whereas Abeta was mainly Abeta-(4-42). In all cases, the presence of Abeta-(X-40) was negligible, a surprising finding in view of the prevalence of Abeta40 in vascular deposits observed in sporadic and familial AD, Down syndrome, and normal aging. Whether the presence of the two amyloid subunits is imperative for the disease phenotype or just reflects a conformational mimicry remains to be elucidated; nonetheless, a specific interaction between ADan oligomers and Abeta molecules was demonstrated in vitro by ligand blot analysis using synthetic peptides. The absence of compact plaques in the presence of extensive neuro fibrillar degeneration strongly suggests that compact plaques, fundamental lesions for the diagnosis of AD, are not essential for the mechanism of dementia
— id: 61252, year: 2005, vol: 280, page: 36883, stat: Journal Article,

Identification of phosphopeptides by MALDI Q-TOF mass spectrometry in positive and negative ion modes after methyl esterification
Xu, Chong-Feng; Lu, Yun; Ma, Jinghong; Mohammadi, Moosa; Neubert, Thomas A
2005 Jun;4(6):809-818, Molecular & cellular proteomics
We have developed an efficient, sensitive and specific method for the detection of phosphopeptides present in peptide mixtures by MALDI Q-TOF mass spectrometry. Use of the MALDI Q-TOF enables selection of phosphopeptides and characterization by collision-induced dissociation of the phosphopeptides performed on the same sample spot. However, this type of experiment has been limited by low ionization efficiency of phosphopeptides in positive ion mode while selecting precursor ions of phosphopeptides. Our method entails neutralizing negative charges on acidic groups of nonphosphorylated peptides by methyl esterification prior to mass spectrometry in positive and negative ion modes. Methyl esterification significantly increases the relative signal intensity generated by phosphopeptides in negative ion mode compared with positive ion mode, and greatly increases selectivity for phosphopeptides by suppressing the signal intensity generated by acidic peptides in negative ion mode. We used the method to identify 12 phosphopeptides containing 22 phosphorylation sites from low femtomolar amounts of a tryptic digest of ss-casein and a-s-casein. We also identified 10 phosphopeptides containing five phosphorylation sites from an in-gel tryptic digest of 100 fmol of an in vitro autophosphorylated fibroblast growth factor receptor kinase domain, and an additional phosphopeptide containing another phosphorylation site when 500 fmol of the digest was examined. The results demonstrate that the method is a fast, robust, and sensitive means of characterizing phosphopeptides present in low abundance mixtures of phosphorylated and nonphosphorylated peptides
— id: 50627, year: 2005, vol: 4, page: 809, stat: Journal Article,

Cleavage of p75 Neurotrophin Receptor by {alpha}-Secretase and {gamma}-Secretase Requires Specific Receptor Domains
Zampieri, Niccolo; Xu, Chong-Feng; Neubert, Thomas A; Chao, Moses V
2005 Apr 15;280(15):14563-14571, Journal of biological chemistry
The p75 neurotrophin receptor (p75(NTR)), a member of the tumor necrosis factor superfamily of receptors, undergoes multiple proteolytic cleavage events. These events are initiated by an alpha-secretase-mediated release of the extracellular domain followed by a gamma-secretase-mediated intramembrane cleavage. However, the specific determinants of p75(NTR) cleavage events are unknown. Many other substrates of gamma-secretase cleavage have been identified, including Notch, amyloid precursor protein, and ErbB4, indicating there is broad substrate recognition by gamma-secretase. Using a series of deletion mutations and chimeric receptors of p75(NTR) and the related Fas receptor, we have identified domains that are essential for p75(NTR) proteolysis. The initial alpha-secretase cleavage was extracellular to the transmembrane domain. Unfortunately, deletion mutants were not capable of defining the requirements of ectodomain shedding. Although this cleavage is promiscuous with respect to amino acid sequence, its position with respect to the transmembrane domain is invariant. The generation of chimeric receptors exchanging different domains of noncleavable Fas receptor with p75(NTR), however, revealed that a discrete domain above the membrane is sufficient for efficient cleavage of p75(NTR). Mass spectrometric analysis confirmed the cleavage can occur with a truncated p75(NTR) displaying only 15 extracellular amino acids in the stalk region
— id: 50628, year: 2005, vol: 280, page: 14563, stat: Journal Article,

Identification and verification of novel rodent postsynaptic density proteins
Jordan, Bryen A; Fernholz, Brian D; Boussac, Muriel; Xu, Chongfeng; Grigorean, Gabriela; Ziff, Edward B; Neubert, Thomas A
2004 Sep;3(9):857-871, Molecular & cellular proteomics
The postsynaptic density (PSD) is a cellular structure specialized in receiving and transducing synaptic information. Here we describe the identification of 452 proteins isolated from biochemically purified PSD fractions of rat and mouse brains using nanoflow HPLC coupled to electrospray tandem mass spectrometry (LC-MS/MS). Fluorescence microscopy and Western blotting were used to verify that many of the novel proteins identified exhibit subcellular distributions consistent with those of PSD-localized proteins. In addition to identifying most previously described PSD components, we also detected proteins involved in signaling to the nucleus as well as regulators of ADP-ribosylation factor signaling, ubiquitination, RNA trafficking, and protein translation. These results suggest new mechanisms by which the PSD helps regulate synaptic strength and transmission
— id: 48196, year: 2004, vol: 3, page: 857, stat: Journal Article,

The N-terminal SH4 region of the Src family kinase Fyn is modified by methylation and heterogeneous fatty acylation: role in membrane targeting, cell adhesion, and spreading
Liang, Xiquan; Lu, Yun; Wilkes, Meredith; Neubert, Thomas A; Resh, Marilyn D
2004 Feb 27;279(9):8133-8139, Journal of biological chemistry
The N-terminal SH4 domain of Src family kinases is responsible for promoting membrane binding and plasma membrane targeting. Most Src family kinases contain an N-terminal Met-Gly-Cys consensus sequence that undergoes dual acylation with myristate and palmitate after removal of methionine. Previous studies of Src family kinase fatty acylation have relied on radiolabeling of cells with radioactive fatty acids. Although this method is useful for verifying that a given fatty acid is attached to a protein, it does not reveal whether other fatty acids or other modifying groups are attached to the protein. Here we use matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry to identify fatty acylated species of the Src family kinase Fyn. Our results reveal that Fyn is efficiently myristoylated and that some of the myristoylated proteins are also heterogeneously S-acylated with palmitate, palmitoleate, stearate, or oleate. Furthermore, we show for the first time that Fyn is trimethylated at lysine residues 7 and/or 9 within its N-terminal region. Both myristoylation and palmitoylation were required for methylation of Fyn. However, a general methylation inhibitor had no inhibitory effect on myristoylation and palmitoylation of Fyn, suggesting that methylation occurs after myristoylation and palmitoylation. Lysine mutants of Fyn that could not be methylated failed to promote cell adhesion and spreading, suggesting that methylation is important for Fyn function
— id: 42155, year: 2004, vol: 279, page: 8133, stat: Journal Article,

ABRF-PRG03: phosphorylation site determination
Arnott, David; Gawinowicz, Mary Ann; Grant, Raymond A; Neubert, Thomas A; Packman, Len C; Speicher, Kaye D; Stone, Kathryn; Turck, Christoph W
2003 Sep;14(3):205-215, Journal of biomolecular techniques
A fundamental aspect of proteomics is the analysis of post-translational modifications, of which phosphorylation is an important class. Numerous nonradioactivity-based methods have been described for high-sensitivity phosphorylation site mapping. The ABRF Proteomics Research Group has conducted a study to help determine how many laboratories are equipped to take on such projects, which methods they choose to apply, and how successful the laboratories are in implementing particular methodologies. The ABRF-PRG03 sample was distributed as a tryptic digest of a mixture of two proteins with two synthetic phosphopeptides added. Each sample contained 5 pmol of unphosphorylated protein digest, 1 pmol of each phosphopeptide from the same protein, and 200 fmol of a minor protein component. Study participants were challenged to identify the two proteins and the two phosphorylated peptides, and determine the site of phosphorylation in each peptide. Almost all respondents successfully identified the major protein component, whereas only 10% identified the minor protein component. Phosphorylation site analysis proved surprisingly difficult, with only 3 of the 54 laboratories correctly determining both sites of phosphorylation. Various strategies and instruments were applied to this task with mixed success; chromatographic separation of the peptides was clearly helpful, whereas enrichment by metal affinity chromatography met with surprisingly little success. We conclude that locating sites of phosphorylation remains a significant challenge at this level of sample abundance
— id: 38150, year: 2003, vol: 14, page: 205, stat: Journal Article,

Facilitated forward chemical genetics using a tagged triazine library and zebrafish embryo screening
Khersonsky, Sonya M; Jung, Da-Woon; Kang, Tae-Wook; Walsh, Daniel P; Moon, Ho-Sang; Jo, Hakryul; Jacobson, Eric M; Shetty, Vivekananda; Neubert, Thomas A; Chang, Young-Tae
2003 Oct 1;125(39):11804-11805, Journal of the American Chemical Society
— id: 38149, year: 2003, vol: 125, page: 11804, stat: Journal Article,

The CD26-related dipeptidyl aminopeptidase-like protein DPPX is a critical component of neuronal A-type K+ channels
Nadal, Marcela S; Ozaita, Andres; Amarillo, Yimy; Vega-Saenz de Miera, Eleazar; Ma, Yuliang; Mo, Wenjun; Goldberg, Ethan M; Misumi, Yoshio; Ikehara, Yukio; Neubert, Thomas A; Rudy, Bernardo
2003 Feb 6;37(3):449-461, Neuron
Subthreshold-activating somatodendritic A-type potassium channels have fundamental roles in neuronal signaling and plasticity which depend on their unique cellular localization, voltage dependence, and kinetic properties. Some of the components of A-type K(+) channels have been identified; however, these do not reproduce the properties of the native channels, indicating that key molecular factors have yet to be unveiled. We purified A-type K(+) channel complexes from rat brain membranes and found that DPPX, a protein of unknown function that is structurally related to the dipeptidyl aminopeptidase and cell adhesion protein CD26, is a novel component of A-type K(+) channels. DPPX associates with the channels' pore-forming subunits, facilitates their trafficking and membrane targeting, reconstitutes the properties of the native channels in heterologous expression systems, and is coexpressed with the pore-forming subunits in the somatodendritic compartment of CNS neurons
— id: 38424, year: 2003, vol: 37, page: 449, stat: Journal Article,

Could TCR antagonism explain associations between MHC genes and disease?
Vukmanovic, Stanislav; Neubert, Thomas A; Santori, Fabio R
2003 Apr;9(4):139-146, Trends in molecular medicine
Alleles of major histocompatibility complex (MHC) loci are associated with certain types of diseases, including those of infectious and autoimmune origin. MHC products can promote susceptibility or resistance to disease by stimulating or inhibiting immune responses. Recent evidence suggests that MHC-associated peptides derived from self-proteins can act as antagonists of T-cell activation, thereby inhibiting immune responses to antigens. We suggest that self-peptide-promoted antagonism might explain some associations between MHC alleles and particular chronic diseases
— id: 34691, year: 2003, vol: 9, page: 139, stat: Journal Article,

Protein-tyrosine Phosphatase-sigma Is a Novel Member of the Functional Family of alpha -Latrotoxin Receptors
Krasnoperov, Valery; Bittner, Mary A; Mo, Wenjun; Buryanovsky, Leonid; Neubert, Thomas A; Holz, Ronald W; Ichtchenko, Konstantin; Petrenko, Alexander G
2002 Sep 27;277(39):35887-35895, Journal of biological chemistry
Receptor-like protein-tyrosine phosphatase sigma (PTPvarsigma) is essential for neuronal development and function. Here we report that PTPvarsigma is a target of alpha-latrotoxin, a strong stimulator of neuronal exocytosis. alpha-Latrotoxin binds to the cell adhesion-like extracellular region of PTPvarsigma. This binding results in the stimulation of exocytosis. The toxin-binding site is located in the C-terminal part of the PTPvarsigma ectodomain and includes two fibronectin type III repeats. The intracellular catalytic domains of PTPvarsigma are not required for the alpha-latrotoxin binding and secretory response triggered by the toxin in chromaffin cells. These features of PTPvarsigma resemble two other previously described alpha-latrotoxin receptors, neurexin and CIRL. Thus, alpha-latrotoxin represents an unusual example of the neurotoxin that has three independent, equally potent, and yet structurally distinct targets. The known structural and functional characteristics of PTPvarsigma, neurexin, and CIRL suggest that they define a functional family of neuronal membrane receptors with complementary or converging roles in presynaptic function via a mechanism that involves cell-to-cell and cell-to-matrix interaction
— id: 32497, year: 2002, vol: 277, page: 35887, stat: Journal Article,

Post-translational proteolytic processing of the calcium-independent receptor of alpha-latrotoxin (CIRL), a natural chimera of the cell adhesion protein and the G protein-coupled receptor. Role of the G protein-coupled receptor proteolysis site (GPS) motif
Krasnoperov, Valery; Lu, Yun; Buryanovsky, Leonid; Neubert, Thomas A; Ichtchenko, Konstantin; Petrenko, Alexander G
2002 Nov 29;277(48):46518-46526, Journal of biological chemistry
The calcium-independent receptor of alpha-latrotoxin (CIRL), a neuronal cell surface receptor implicated in the regulation of exocytosis, is a natural chimera of the cell adhesion protein and the G protein-coupled receptor (GPCR). In contrast with canonic GPCRs, CIRL consists of two heterologous non-covalently bound subunits, p120 and p85, due to endogenous proteolytic processing of the receptor precursor in the endoplasmic reticulum. Extracellularly oriented p120 contains hydrophilic cell adhesion domains, whereas p85 resembles a generic GPCR. We determined that the site of the CIRL cleavage is located within a juxtamembrane Cys- and Trp-rich domain of the N-terminal extracellular region of CIRL. Mutations in this domain make CIRL resistant to the cleavage and impair its trafficking. Therefore, we have named it GPS for G protein-coupled receptor proteolysis site. The GPS motif is found in homologous adhesion GPCRs and thus defines a novel receptor family. We postulate that the proteolytic processing and two-subunit structure is a common characteristic feature in the family of GPS-containing adhesion GPCRs
— id: 33173, year: 2002, vol: 277, page: 46518, stat: Journal Article,

Mass Spectrometric Analysis of GAP-43/Neuromodulin Reveals the Presence of a Variety of Fatty Acylated Species
Liang, Xiquan; Lu, Yun; Neubert, Thomas A; Resh, Marilyn D
2002 Sep 6;277(36):33032-33040, Journal of biological chemistry
GAP-43 (neuromodulin) is a protein kinase C substrate that is abundant in developing and regenerating neurons. Thioester-linked palmitoylation at two cysteines near the GAP-43 N terminus has been implicated in directing membrane binding. Here, we use mass spectrometry to examine the stoichiometry of palmitoylation and the molecular identity of the fatty acid(s) attached to GAP-43 in vivo. GAP-43 expressed in either PC12 or COS-1 cells was acetylated at the N-terminal methionine. Approximately 35% of the N-terminal GAP-43 peptides were also modified by palmitate and/or stearate on Cys residues. Interestingly, a variety of acylated species was detected, in which one of the Cys residues was acylated by either palmitate or stearate, or both Cys residues were acylated by palmitates or stearates or a combination of palmitate and stearate. Depalmitoylation of membrane-bound GAP-43 did not release the protein from the membrane, implying that additional forces function to maintain membrane binding. Indeed, mutation of four basic residues within the N-terminal domain of GAP-43 dramatically reduced membrane localization of GAP-43 without affecting palmitoylation. These data reveal the heterogeneous nature of S-acylation in vivo and illustrate the power of mass spectrometry for identification of key regulatory protein modifications
— id: 32498, year: 2002, vol: 277, page: 33032, stat: Journal Article,

Rare, structurally homologous self-peptides promote thymocyte positive selection
Santori, Fabio R; Kieper, William C; Brown, Stuart M; Lu, Yun; Neubert, Thomas A; Johnson, Kenneth L; Naylor, Stephen; Vukmanovic, Stanislav; Hogquist, Kristin A; Jameson, Stephen C
2002 Aug;17(2):131-142, Immunity
Although it is clear that positive selection of T cells involves recognition of specific self-peptide/MHC complexes, the nature of these self-ligands and their relationship to the cognate antigen are controversial. Here we used two complementary strategies to identify naturally occurring self-peptides able to induce positive selection of T cells bearing a specific T cell receptor, OT-I. Both the bioassay- and bioinformatics-based strategies identified the same self-peptides, derived from F-actin capping protein and beta-catenin. These peptides displayed charge conservation at two key TCR contact residues. The biological activity of 43 other self-peptides and of complex peptide libraries directly correlated to the extent of conservation at TCR contact residues. These results demonstrate that selecting self-peptides are rare and can be identified by homology-based search strategies
— id: 32496, year: 2002, vol: 17, page: 131, stat: Journal Article,

Crystal Structure of the MuSK Tyrosine Kinase. Insights into Receptor Autoregulation
Till, Jeffrey H; Becerra, Manuel; Watty, Anke; Lu, Yun; Ma, Yuliang; Neubert, Thomas A; Burden, Steven J; Hubbard, Stevan R
2002 Sep;10(9):1187-1187, Structure
Muscle-specific kinase (MuSK) is a receptor tyrosine kinase expressed selectively in skeletal muscle. During neuromuscular synapse formation, agrin released from motor neurons stimulates MuSK autophosphorylation in the kinase activation loop and in the juxtamembrane region, leading to clustering of acetylcholine receptors. We have determined the crystal structure of the cytoplasmic domain of unphosphorylated MuSK at 2.05 A resolution. The structure reveals an autoinhibited kinase domain in which the activation loop obstructs ATP and substrate binding. Steady-state kinetic analysis demonstrates that autophosphorylation results in a 200-fold increase in k(cat) and a 10-fold decrease in the K(m) for ATP. These studies provide a molecular basis for understanding the regulation of MuSK catalytic activity and suggest that an additional in vivo component may contribute to regulation via the juxtamembrane region
— id: 32906, year: 2002, vol: 10, page: 1187, stat: Journal Article,

Definitive Identification of Mammalian 5-Hydroxymethyluracil DNA N-Glycosylase Activity as SMUG1
Boorstein RJ; Cummings A Jr; Marenstein DR; Chan MK; Ma Y; Neubert TA; Brown SM; Teebor GW
2001 Nov 9;276(45):41991-41997, Journal of biological chemistry
Purification from calf thymus of a DNA N-glycosylase activity (HMUDG) that released 5-hydroxymethyluracil (5hmUra) from the DNA of Bacillus subtilis phage SPO1 was undertaken. Analysis of the most purified fraction by SDS-polyacrylamide gel electrophoresis revealed a multiplicity of protein species making it impossible to identify HMUDG by inspection. Therefore, we renatured the enzyme after SDS-polyacrylamide gel electrophoresis and assayed slices of the gel for DNA N-glycosylase activity directed against 5hmUra. Maximum enzymatic activity was identified between molecular mass markers 30 and 34 kDa. Protein was extracted from gel slices and subjected to tryptic digestion and analysis by mass spectrometry. Analysis revealed the presence of 11 peptides that were homologous or identical to the sequence of the recently characterized human single-stranded monofunctional uracil DNA N-glycosylase (hSMUG1). The cDNA of hSMUG1 was isolated and expressed as a recombinant glutathione S-transferase fusion protein that was shown to release 5hmUra with 20x the specific activity of the most purified bovine fraction. We conclude that hSMUG1 and HMUDG are the same protein
— id: 23928, year: 2001, vol: 276, page: 41991, stat: Journal Article,

Systemic Amyloid Deposits in Familial British Dementia
Ghiso JA; Holton J; Miravalle L; Calero M; Lashley T; Vidal R; Houlden H; Wood N; Neubert TA; Rostagno A; Plant G; Revesz T; Frangione B
2001 Nov 23;276(47):43909-43914, Journal of biological chemistry
Familial British dementia (FBD) is an early onset inherited disorder that, like familial Alzheimer's disease (FAD), is characterized by progressive dementia, amyloid deposition in the brain, and neurofibrillary degeneration of limbic neurons. The primary structure of the amyloid subunit (ABri) extracted from FBD brain tissues (Vidal, R., Frangione, B., Rostagno, A., Mead, S., Revesz, T., Plant, G., and Ghiso, J. (1999) Nature 399, 776-781) is entirely different and unrelated to any previously known amyloid protein. Patients with FBD have a single nucleotide substitution at codon 267 in the BRI2 gene, resulting in an arginine replacing the stop codon and a longer open reading frame of 277 amino acids instead of 266. The ABri peptide comprises the 34 C-terminal residues of the mutated precursor ABriPP-277 and is generated via furin-like proteolytic processing. Here we report that carriers of the Stop-to-Arg mutation have a soluble form of the amyloid peptide (sABri) in the circulation with an estimated concentration in the range of 20 ng/ml, several fold higher than that of soluble Abeta. In addition, ABri species identical to those identified in the brain were also found as fibrillar components of amyloid deposits predominantly in the blood vessels of several peripheral tissues, including pancreas and myocardium. We hypothesize that the high concentration of the soluble de novo created amyloidogenic peptide and/or the insufficient tissue clearance are the main causative factors for the formation of amyloid deposits outside the brain. Thus, FBD constitutes the first documented cerebral amyloidosis associated with neurodegeneration and dementia in which the amyloid deposition is also systemic
— id: 23926, year: 2001, vol: 276, page: 43909, stat: Journal Article,

Characterization of phosphopeptides from protein digests using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nanoelectrospray quadrupole time-of-flight mass spectrometry
Ma Y; Lu Y; Zeng H; Ron D; Mo W; Neubert TA
2001 ;15(18):1693-1700, Rapid communications in mass spectrometry
A two-step mass spectrometric method for characterization of phosphopeptides from peptide mixtures is presented. In the first step, phosphopeptide candidates were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) based on their higher relative intensities in negative ion MALDI spectra than in positive ion MALDI spectra. The detection limit for this step was found to be 18 femtomoles or lower in the case of unfractionated in-solution digests of a model phosphoprotein, beta-casein. In the second step, nanoelectrospray tandem mass (nES-MS/MS) spectra of doubly or triply charged precursor ions of these candidate phosphopeptides were obtained using a quadrupole time-of-flight (Q-TOF) mass spectrometer. This step provided information about the phosphorylated residues, and ruled out nonphosphorylated candidates, for these peptides. After [(32)P] labeling and reverse-phase high-performance liquid chromatography (RP-HPLC) to simplify the mixtures and to monitor the efficiency of phosphopeptide identification, we used this method to identify multiple autophosphorylation sites on the PKR-like endoplasmic reticulum kinase (PERK), a recently discovered mammalian stress-response protein
— id: 23927, year: 2001, vol: 15, page: 1693, stat: Journal Article,

Cutting Edge: Positive Selection Induced by a Self-Peptide with TCR Antagonist Activity
Santori FR; Brown SM; Lu Y; Neubert TA; Vukmanovic S
2001 Dec 1;167(11):6092-6095, Journal of immunology
Antagonist-like engagement of the TCR has been proposed to induce T cell selection in the thymus. However, no natural TCR ligand with TCR antagonist activity is presently known. Using a combination of bioinformatics and functional testing we identified the first self-peptide that can both deliver antagonist-like signals and promote T cell selection in the thymus. The peptide is presented by appropriate MHC class I molecules in vivo. Thus, endogenous antagonist peptides exist and may be involved in TCR repertoire selection
— id: 23925, year: 2001, vol: 167, page: 6092, stat: Journal Article,

Uroplakin Ia is the urothelial receptor for uropathogenic Escherichia coli: evidence from in vitro FimH binding
Zhou G; Mo WJ; Sebbel P; Min G; Neubert TA; Glockshuber R; Wu XR; Sun TT; Kong XP
2001 Nov;114(Pt 22):4095-4103, Journal of cell science
The binding of uropathogenic Escherichia coli to the urothelial surface is a crucial initial event for establishing urinary tract infection because it allows the bacteria to gain a foothold on the urothelial surface, thus preventing them from being removed by micturition. In addition, it triggers bacterial invasion as well as host urothelial defense. This binding is mediated by the FimH adhesin located at the tip of the bacterial type 1-fimbrium, a filamentous attachment apparatus, and its urothelial receptor. We have prepared a biotinylated, recombinant FimH-FimC adhesin:chaperone complex and used it to identify its mouse urothelial receptor. The FimH-FimC complex binds specifically to a single 24 kDa major mouse urothelial plaque protein, which we identified as uroplakin Ia by mass spectrometry, cDNA cloning and immunoreactivity. The terminal mannosyl moieties on Asn-169 of uroplakin Ia are responsible for FimH as well as concanavalin A binding. Although FimH binds to uroplakin Ia with only moderate strength (K(d) approximately 100 nM between pH 4 and 9), the binding between multiple fimbriae of a bacterium and the crystalline array of polymerized uroplakin receptors should achieve high avidity and stable bacterial attachment. The FimH-FimC complex binds preferentially to the mouse urothelial umbrella cells in a pattern similar to uroplakin staining. Our results indicate that the structurally related uroplakins Ia and Ib are glycosylated differently, that uroplakin Ia serves as the urothelial receptor for the type 1-fimbriated E. coli, and that the binding of uropathogenic bacteria to uroplakin Ia may play a key role in mediating the urothelial responses to bacterial attachment
— id: 26903, year: 2001, vol: 114, page: 4095, stat: Journal Article,

Sequencing of oxidized methionine-containing peptides for protein identification
Mo W; Ma Y; Takao T; Neubert TA
2000 ;14(21):2080-2081, Rapid communications in mass spectrometry
— id: 23929, year: 2000, vol: 14, page: 2080, stat: Journal Article,