Stable isotope labeling with amino acids in Drosophila for quantifying proteins and modifications.

Drosophila melanogaster is a common animal model for genetics studies, and quantitative proteomics studies of the fly are emerging. Here, we present in detail the development of a procedure to incorporate stable isotope-labeled amino acids into the fly proteome. In the method of stable isotope labeling with amino acids in Drosophila melanogaster (SILAC fly), flies were fed with SILAC-labeled yeast grown with modified media, enabling near complete labeling in a single generation.

Optically switchable transistor via energy-level phototuning in a bicomponent organic semiconductor.

Organic semiconductors are suitable candidates for printable, flexible and large-area electronics. Alongside attaining an improved device performance, to confer a multifunctional nature to the employed materials is key for organic-based logic applications. Here we report on the engineering of an electronic structure in a semiconducting film by blending two molecular components, a photochromic diarylethene derivative and a poly(3-hexylthiophene) (P3HT) matrix, to attain phototunable and bistable energy levels for the P3HT's hole transport.

Tissue-type plasminogen activator regulates the neuronal uptake of glucose in the ischemic brain.

The ability to sense and adapt to hypoxic conditions plays a pivotal role in neuronal survival. Hypoxia induces the release of tissue-type plasminogen activator (tPA) from cerebral cortical neurons. We found that the release of neuronal tPA or treatment with recombinant tPA promotes cell survival in cerebral cortical neurons previously exposed to hypoxic conditions in vitro or experimental cerebral ischemia in vivo.

Coaggregation of RNA-binding proteins in a model of TDP-43 proteinopathy with selective RGG motif methylation and a role for RRM1 ubiquitination.

TAR DNA-binding protein 43 (TDP-43) is a major component within ubiquitin-positive inclusions of a number of neurodegenerative diseases that increasingly are considered as TDP-43 proteinopathies. Identities of other inclusion proteins associated with TDP-43 aggregation remain poorly defined. In this study, we identify and quantitate 35 co-aggregating proteins in the detergent-resistant fraction of HEK-293 cells in which TDP-43 or a particularly aggregate prone variant, TDP-S6, were enriched following overexpression, using stable isotope-labeled (SILAC) internal standards and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS).

Asparaginyl endopeptidase cleaves TDP-43 in brain.

TAR DNA-binding protein 43 (TDP-43) is a nuclear protein involved in RNA splicing and a major protein component in ubiquitin-positive, tau-negative inclusions of frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Under disease conditions, TDP-43 redistributes to the cytoplasm where it can be phosphorylated, ubiquitinated, and proteolytically cleaved. Enzymes responsible for TDP-43 proteolytic processing in brain remain largely unreported.

Protein Cysteines Map to Functional Networks According to Steady-state Level of Oxidation.

The cysteine (Cys) proteome serves critical roles in protein structure, function and regulation, and includes key targets in oxidative mechanisms of disease. Thioredoxins maintain Cys residues in thiol forms, and previous research shows that the redox potential of thioredoxin in mitochondria and nuclei is more reduced than cytoplasm, suggesting that proteins in these compartments may have different steady-state oxidation. This study measured fractional oxidation of 641 peptidyl Cys residues from 333 proteins in HT29 cells by mass spectrometry.

Analysis of a membrane-enriched proteome from postmortem human brain tissue in Alzheimer's disease.

Purpose: The present study is a discovery mode proteomics analysis of the membrane-enriched fraction of postmortem brain tissue from Alzheimer's disease (AD) and control cases. This study aims to validate a method to identify new proteins that could be involved in the pathogenesis of AD and potentially serve as disease biomarkers.

Experimental Design: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to analyze the membrane-enriched fraction of human postmortem brain tissue from five AD and five control cases of similar age.

Quantitative analysis of the detergent-insoluble brain proteome in frontotemporal lobar degeneration using SILAC internal standards.

A hallmark of neurodegeneration is the aggregation of disease related proteins that are resistant to detergent extraction. In the major pathological subtype of frontotemporal lobar degeneration (FTLD), modified TAR-DNA binding protein 43 (TDP-43), including phosphorylated, ubiquitinated, and proteolytically cleaved forms, is enriched in detergent-insoluble fractions from post-mortem brain tissue. Additional proteins that accumulate in the detergent-insoluble FTLD brain proteome remain largely unknown.

Aberrant septin 11 is associated with sporadic frontotemporal lobar degeneration.

Background: Detergent-insoluble protein accumulation and aggregation in the brain is one of the pathological hallmarks of neurodegenerative diseases. Here, we describe the identification of septin 11 (SEPT11), an enriched component of detergent-resistant fractions in frontotemporal lobar degeneration with ubiquitin-immunoreactive inclusions (FTLD-U), using large-scale unbiased proteomics approaches.

Results: We developed and applied orthogonal quantitative proteomic strategies for the unbiased identification of disease-associated proteins in FTLD-U.

Implementing knowledge into practice for improved neonatal survival; a cluster-randomised, community-based trial in Quang Ninh province, Vietnam.

Background: Globally, almost 4 million newborns die during the first 4 weeks of life every year. By increased use of evidence-based knowledge in the healthcare system a large proportion of these neonatal deaths could be prevented. But there is a severe lack of knowledge on effective methods for successful implementation of evidence into practice, particularly in low- and middle-income countries.

Proteomic analysis of hippocampal dentate granule cells in frontotemporal lobar degeneration: application of laser capture technology.

Frontotemporal lobar degeneration (FTLD) is the most common cause of dementia with pre-senile onset, accounting for as many as 20% of cases. A common subset of FTLD cases is characterized by the presence of ubiquitinated inclusions in vulnerable neurons (FTLD-U). While the pathophysiological mechanisms underlying neurodegeneration in FTLD-U have not yet been elucidated, the presence of inclusions in this disease indicates enhanced aggregation of one or several proteins.

Polyubiquitin linkage profiles in three models of proteolytic stress suggest the etiology of Alzheimer disease.

Polyubiquitin chains on substrates are assembled through any of seven lysine residues or the N terminus of ubiquitin (Ub), generating diverse linkages in the chain structure. PolyUb linkages regulate the fate of modified substrates, but their abundance and function in mammalian cells are not well studied. We present a mass spectrometry-based method to measure polyUb linkages directly from total lysate of mammalian cells.

Phosphoproteomic analysis reveals site-specific changes in GFAP and NDRG2 phosphorylation in frontotemporal lobar degeneration.

Frontotemporal lobar degeneration (FTLD) is a progressive neurodegenerative disease characterized by behavioral abnormalities, personality changes, language dysfunction, and can co-occur with the development of motor neuron disease. One major pathological form of FTLD is characterized by intracellular deposition of ubiquitinated and phosphorylated TAR DNA binding protein-43 (TDP-43), suggesting that dysregulation in phosphorylation events may contribute to disease progression. However, to date systematic analysis of the phosphoproteome in FTLD brains has not been reported.

Galectin-3 is a candidate biomarker for amyotrophic lateral sclerosis: discovery by a proteomics approach.

The discovery of biomarkers for neurodegenerative diseases will have a major impact on the efficiency of therapeutic clinical trials and may be important for understanding basic pathogenic mechanisms. We have approached the discovery of protein biomarkers for amyotrophic lateral sclerosis (ALS) by profiling affected tissues in a relevant animal model and then validating the findings in human tissues. Ventral roots from SOD1(G93A) "ALS" mice were analyzed by label-free quantitative mass spectrometry, and the resulting data were compared with data for matched samples from nontransgenic littermates and transgenic mice carrying wild-type human SOD1 (SOD1(WT)).

Multiplex SILAC analysis of a cellular TDP-43 proteinopathy model reveals protein inclusions associated with SUMOylation and diverse polyubiquitin chains.

Transactive response (TAR) DNA-binding protein 43 (TDP-43) is a major protein component within ubiquitin-positive inclusions of frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Although TDP-43 is a nuclear DNA/RNA-binding protein, in pathological conditions, TDP-43 has been reported to redistribute to the cytoplasm where it is cleaved and forms insoluble, ubiquitinated, and phosphorylated inclusions. Here we present a cellular model in which full-length human TDP-43 or a splicing isoform (TDP-S6) that lacks the C terminus is overexpressed in a human cell line and mouse primary neurons.

Proteomics analysis reveals novel components in the detergent-insoluble subproteome in Alzheimer's disease.

Neurodegenerative diseases are often defined pathologically by the presence of protein aggregates. These aggregates, including amyloid plaques in Alzheimer's disease (AD), result from the abnormal accumulation and processing of proteins, and may ultimately lead to neuronal dysfunction and cell death. To date, conventional biochemical studies have revealed abundant core components in protein aggregates.

Systematical optimization of reverse-phase chromatography for shotgun proteomics.

We report the optimization of a common LC-MS/MS platform to maximize the number of proteins identified from a complex biological sample. The platform uses digested yeast lysate on a 75 microm internal diameter x 12 cm reverse-phase column that is combined with an LTQ-Orbitrap mass spectrometer. We first generated a yeast peptide mix that was quantified by multiple methods including the strategy of stable isotope labeling with amino acids in cell culture (SILAC).

Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation.

All seven lysine residues in ubiquitin contribute to the synthesis of polyubiquitin chains on protein substrates. Whereas K48-linked chains are well established as mediators of proteasomal degradation, and K63-linked chains act in nonproteolytic events, the roles of unconventional polyubiquitin chains linked through K6, K11, K27, K29, or K33 are not well understood. Here, we report that the unconventional linkages are abundant in vivo and that all non-K63 linkages may target proteins for degradation.

Identification and characterization of neuronal mitogen-activated protein kinase substrates using a specific phosphomotif antibody.

Mitogen-activated protein kinases (MAPKs) control neuronal synaptic function; however, little is known about the synaptic substrates regulated by MAPKs. A phosphopeptide library incorporating the MAPK consensus motif (PX(pS/pT)P where pS is phosphoserine and pT is phosphothreonine) was used to raise a phosphospecific antibody that detected MAPK-mediated phosphorylation. The antibody (termed "5557") recognized a variety of phosphoproteins in the brain, many of which were enriched in postsynaptic density fractions.

Phosphoproteomic analysis of human brain by calcium phosphate precipitation and mass spectrometry.

Alzheimer's disease (AD), the most common form of dementia, is manifested in the brain by the aggregation of amyloid plaques and neurofibrillary tangles. The tangles are primarily composed of microtubule-associated protein tau that is aberrantly hyperphosphorylated, suggesting that deregulated phosphorylation may contribute to AD pathogenesis. However, systematic analysis of the phosphoproteome in AD brain tissues has not been reported.

Proteomic identification of novel proteins associated with Lewy bodies.

The manifestation of Lewy bodies (LB) in the brain is a hallmark of Parkinson's disease. Here, we present a comprehensive analysis of protein elements in Lewy bodies by comparative mass spectrometry. Cortical LB inclusions were enriched by sucrose gradient centrifugation from postmortem brains, and a negative control sample was prepared from specimen without LB pathology.

Systematic approach for validating the ubiquitinated proteome.

Protein ubiquitination plays an essential regulatory role within all eukaryotes. Large-scale analyses of ubiquitinated proteins are usually performed by combining affinity purification strategies with mass spectrometry. However, there is no reliable method to systematically differentiate ubiquitinated species from copurified unmodified components.

Neuronal morphogenesis is regulated by the interplay between cyclin-dependent kinase 5 and the ubiquitin ligase mind bomb 1.

Neuronal communication requires the coordinated assembly of polarized structures including axons, dendrites, and synapses. Here, we report the identification of a ubiquitin ligase mind bomb 1 (Mib1) in the postsynaptic density and the characterization of its role in neuronal morphogenesis. Expression of Mib1 inhibits neurite outgrowth in cell culture and its gene deletion enhances synaptic growth at the neuromuscular junction in Drosophila.

Merger of laser capture microdissection and mass spectrometry: a window into the amyloid plaque proteome.

The occurrence of protein accumulation and aggregation in the brain is one of the pathological hallmarks of neurodegenerative diseases such as Alzheimer's disease (AD). Although it is instructive to analyze the aggregated proteins in the brain, biochemical purification and identification of these proteins have been challenging. Recent developments in laser capture microdissection (LCM) and mass spectrometry (MS) enable large-scale protein profiling of captured tissue samples.

Relative and absolute quantification of postsynaptic density proteome isolated from rat forebrain and cerebellum.

The postsynaptic density (PSD) of central excitatory synapses is essential for postsynaptic signaling, and its components are heterogeneous among different neuronal subtypes and brain structures. Here we report large scale relative and absolute quantification of proteins in PSDs purified from adult rat forebrain and cerebellum. PSD protein profiles were determined using the cleavable ICAT strategy and LC-MS/MS.