Martin Hornshaw

Thermo Fisher Scientific, Waltham, Massachusetts, United States

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Publications (23)88.24 Total impact

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    Martin Hornshaw
    03/2014; 2:63–64. DOI:10.1016/j.euprot.2013.11.006
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    ABSTRACT: Cerebrospinal fluid (CSF) biomarkers for Alzheimer's disease (AD) are increasingly used in research centers, clinical trials, and clinical settings. However, their broad-scale use is hampered by lack of standardization across analytical platforms and by interference from binding of amyloid-β (Aβ) to matrix proteins as well as self-aggregation. Here, we report on a matrix effect-resistant method for the measurement of the AD-associated 42 amino acid species of Aβ (Aβ42), together with Aβ40 and Aβ38 in human CSF based on mass spectrometric quantification using selected reaction monitoring (SRM). Samples were prepared by solid-phase extraction and quantification was performed using stable-isotope labeled Aβ peptides as internal standards. The diagnostic performance of the method was evaluated on two independent clinical materials with research volunteers who were cognitively normal and AD patients with mild to moderate dementia. Analytical characteristics of the method include a lower limit of quantification of 62.5 pg/mL for Aβ42 and coefficients of variations below 10%. In a pilot study on AD patients and controls, we verified disease-association with decreased levels of Aβ42 similar to that obtained by ELISA and even better separation was obtained using the Aβ42/Aβ40 ratio. The developed assay is sensitive and is not influenced by matrix effects, enabling absolute quantification of Aβ42, Aβ40, and Aβ38 in CSF, while it retains the ability to distinguish AD patients from controls. We suggest this SRM-based method for Aβ peptide quantification in human CSF valuable for clinical research and trials.
    Journal of Alzheimer's disease: JAD 10/2012; 33(4). DOI:10.3233/JAD-2012-121471 · 3.61 Impact Factor
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    ABSTRACT: The degree of precision in measuring accurate masses in LC MS/MS-based metabolomics experiments is a determinant in the successful identification of the metabolites present in the original extract. Using the methods described here, complex broccoli extracts containing hundreds of small-molecule compounds (mass range 100-1,400 Da) can be profiled at resolutions up to 100,000 (full width half maximum, FWHM), useful for accurate and sensitive relative quantification experiments. Using external instrument calibration, analyte masses can be measured with high (sub-ppm to a maximum of 2 ppm) accuracy, leading to compound identifications based on elemental composition analysis. Unambiguous identification of four analytes (citric acid, chlorogenic acid, phenylalanine, and UDP-D: -glucose) is used to validate the performance of the different MS/MS fragmentation regimes. Identifications are carried out either via resonance excitation collision induced dissociation (CID) or via higher energy collision dissociation (HCD) experiments, and validated by infrared multiphoton dissociation (IRMPD) fragmentation of standards. Such results, obtained on both hybrid and non-hybrid systems from metabolite profiling and identification experiments, provide evidence that the strategies selected can be successfully applied to other LC-MS based projects for plant metabolomic studies.
    Methods in molecular biology (Clifton, N.J.) 01/2012; 860:145-56. DOI:10.1007/978-1-61779-594-7_10 · 1.29 Impact Factor
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    ABSTRACT: Amphibian skin secretions are, for the most part, complex peptidomes. While many peptide components have been biologically- and structurally-characterised into discrete "families", some of which are analogues of endogenous vertebrate regulatory peptides, a substantial number are of unique structure and unknown function. Among the components of these secretory peptidomes is an array of protease inhibitors. Inhibitors of trypsin are of widespread occurrence in different taxa and are representative of many established structural classes, including Kunitz, Kazal and Bowman-Birk. However, few protease inhibitors with activity against other specific proteases have been described from this source. Here we report for the first time, the isolation and structural characterisation of an inhibitor of chymotrypsin of Kunitz-type from the skin secretion of the African hyperoliid frog, Kassina senegalensis. To this end, we employed a functional peptidomic approach. This scheme involves fractionation of the peptidome, functional end-point screening, structural characterisation of resultant actives followed by molecular cloning of biosynthetic precursor-encoding cDNA(s). The novel mature and active polypeptide identified consisted of 62 amino acid residues (average molecular mass 6776.24 Da), of which 6 were positionally-conserved cysteines. The P(1) position within the active site was occupied by a phenylalanyl residue. Bioinformatic analysis of the sequence using BLAST, revealed a structural similarity to Kunitz-type chymotrypsin inhibitors from other organisms, ranging from silkworms to snakes.
    Biochimie 12/2011; 94(3):891-9. DOI:10.1016/j.biochi.2011.12.008 · 3.14 Impact Factor
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    ABSTRACT: This article provides an introduction to Fourier transform-based mass spectrometry. The key performance characteristics of Fourier transform-based mass spectrometry, mass accuracy and resolution, are presented in the view of how they impact the interpretation of measurements in proteomic applications. The theory and principles of operation of two types of mass analyzer, Fourier transform ion cyclotron resonance and Orbitrap, are described. Major benefits as well as limitations of Fourier transform-based mass spectrometry technology are discussed in the context of practical sample analysis, and illustrated with examples included as figures in this text and in the accompanying slide set. Comparisons highlighting the performance differences between the two mass analyzers are made where deemed useful in assisting the user with choosing the most appropriate technology for an application. Recent developments of these high-performing mass spectrometers are mentioned to provide a future outlook.
    Molecular &amp Cellular Proteomics 07/2011; 10(7):M111.009431. DOI:10.1074/mcp.M111.009431 · 7.25 Impact Factor
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    ABSTRACT: Amphibian skin secretions are established sources of bioactive peptides. Here we describe the isolation, structural and pharmacological characterisation of a novel vasoconstrictor peptide from the skin secretion of the African hyperoliid frog, Kassina maculata, which exhibits no structural similarity to any known class of amphibian skin peptide. The peptide consists of 21 amino acid residues, FIKELLPHLSGIIDSVANAIK, and is C-terminally amidated. The provisional structure was obtained by MS/MS fragmentation using an Orbitrap mass spectrometer and L/I ambiguities were resolved following molecular cloning of biosynthetic precursor-encoding cDNA. A synthetic replicate of the peptide was found to possess weak antimicrobial and haemolytic activities but was exceptionally effective in constricting the smooth muscle of rat tail artery (EC(50) of 25 pM). In reflection of its exceptional potency in constricting rat arterial smooth muscle, the peptide was named kasstasin, a derivation of Kassina and "stasis" (stoppage of flow). These data illustrate the continuing potential of amphibian skin secretions to provide novel natural peptide templates for biological evaluation.
    Biochimie 05/2011; 93(9):1537-42. DOI:10.1016/j.biochi.2011.05.009 · 3.14 Impact Factor
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    ABSTRACT: This article provides an introduction to Fourier transform-based mass spectrometry (FTMS). The key performance characteristics of FTMS, mass accuracy and resolution, are presented in the view of how they impact the interpretation of measurements in proteomic applications. The theory and principles of operation of two types of mass analyzer, Fourier transform ion cyclotron resonance and Orbitrap, are described. Major benefits as well as limitations of FTMS technology are discussed in the context of practical sample analysis, and illustrated with examples included as figures in this text and in the accompanying slide set. Comparisons highlighting the performance differences between the two mass analyzers are made where deemed useful in assisting the user with choosing the most appropriate technology for his/her application. Recent developments of these high-performing mass spectrometers are mentioned to provide a future outlook.
    Molecular &amp Cellular Proteomics 05/2011; DOI:10.1074/mcp.O111.009431 · 7.25 Impact Factor
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    ABSTRACT: Products for specific diagnosis and immunotherapy of IgE-mediated allergies are currently based on natural extracts. Quantification of major allergen content is an important aspect of standardization as important allergens particularly impact vaccine potency. The aim of the study was to develop a mass spectrometry (MS) based assay for absolute quantification of Timothy (Phleum pratense) pollen allergens Phl p 1 and Phl p 5 in P. pratense extract. High-resolution and accurate mass (HRAM) MS was selected for its ability to detect peptides with high selectivity and mass accuracy (<3 ppm). Isotope labeled heavy peptides were used for absolute quantification of specific isoallergens of Phl p 1 and Phl p 5 at low femtomole level in P. pratense extract. Robustness and linearity of the method was demonstrated with intra day precision ≤ 5% (n = 3). Phl p 1b was shown to be 5 times less abundant than its variant Phl p 1a and Phl p 5b was shown to be 9 times more abundant than the Phl p 5a. The present study shows that allergen, and/or isoallergen specific, surrogate signature peptides analyzed with HRAM MS is a sensitive and accurate tool for identification and quantification of allergens from complex allergen sources.
    Journal of Proteome Research 02/2011; 10(4):2113-22. DOI:10.1021/pr101150z · 5.06 Impact Factor
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    ABSTRACT: From defensive skin secretions acquired from two species of African hyperoliid frogs, Kassina maculata and Kassina senegalensis, we have isolated two structurally related, C-terminally amidated tridecapeptides of novel primary structure that exhibit a broad spectrum of biological activity. In reflection of their structural novelty and species of origin, we named the peptides kassorin M (FLEGLLNTVTGLLamide; 1387.8 Da) and kassorin S (FLGGILNTITGLLamide; 1329.8 Da), respectively. The primary structure and organisation of the biosynthetic precursors of kassorins M and S were deduced from cloned skin secretion-derived cDNA. Both open-reading frames encoded a single copy of kassorin M and S, respectively, located at the C-terminus. Kassorins display limited structural similarities to vespid chemotactic peptides (7/13 residues), temporin A (5/13 residues), the N-terminus of Lv-ranaspumin, a foam nest surfactant protein of the frog, Leptodactylus vastus, and an N-terminal domain of the equine sweat surfactant protein, latherin. Both peptides elicit histamine release from rat peritoneal mast cells. However, while kassorin S was found to possess antibacterial activity against Staphylococcus aureus, kassorin M was devoid of such activity. In contrast, kassorin M was found to contract the smooth muscle of guinea pig urinary bladder (EC(50) = 4.66 nM) and kassorin S was devoid of this activity. Kassorins thus represent the prototypes of a novel family of peptides from the amphibian innate immune system as occurring in defensive skin secretions.
    Molecular Immunology 10/2010; 48(4):442-51. DOI:10.1016/j.molimm.2010.09.018 · 3.00 Impact Factor
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    ABSTRACT: In this study two regions of embryonic (E11) mouse central nervous system (CNS) have been profiled for their unesterified sterol content. Using high-performance liquid chromatography (HPLC)-mass spectrometry (MS) and tandem mass spectrometry (MS(n)) low levels of oxysterols (estimated 2-165 ng g(-1) wet weight) were identified in cortex (Ctx) and spinal cord (Sc). The identified oxysterols include 7 alpha-, 7 beta-, 22R-, 24S-, 25- and 27-hydroxycholesterol; 24,25- and 24,27-dihydroxycholesterol; and 24S,25-epoxycholesterol. Of these, 24S-hydroxycholesterol is biosynthesised exclusively in brain. In comparison to adult mouse where the 24S-hydroxycholesterol level is about 40 microg g(-1) in brain the level of 24S-hydroxycholesterol reported here (estimated 26 ng g(-1) in Ctx and 13 ng g(-1) in Sc) is extremely low. Interestingly, the level of 24S,25-epoxycholesterol in both CNS regions (estimated 165 ng g(-1) in Ctx and 91 ng g(-1) in Sc) is somewhat higher than the levels of the hydroxycholesterols. This oxysterol is formed in parallel to cholesterol via a shunt of the mevalonate pathway and its comparatively high abundance may be a reflection of a high rate of cholesterol synthesis at this stage of development. Levels of cholesterol (estimated 1.25 mg g(-1) in Ctx and 1.15 mg g(-1) in Sc) and its precursors were determined by gas chromatography-mass spectrometry (GC-MS). In both CNS regions cholesterol levels were found to be lower than those reported in the adult, but in relation to cholesterol the levels of cholesterol precursors were higher than found in adult indicating a high rate of cholesterol synthesis. In summary, our data provide evidence for the presence of endogenous oxysterols in two brain regions of the developing CNS. Moreover, while most of the enzymes involved in hydroxysterol synthesis are minimally active at E11, our results suggest that the mevalonate pathway is significantly active, opening up the possibility for a function of 24S,25-epoxycholesterol during brain development.
    Molecular BioSystems 06/2009; 5(5):529-41. DOI:10.1039/b819502a · 3.35 Impact Factor
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    ABSTRACT: While the proteome defines the expressed gene products, the metabolome results from reactions controlled by such gene products. Plasma represents an accessible "window" to the metabolome both in regard of availability and content. The wide range of the plasma metabolome, in terms of molecular diversity and abundance, makes its comprehensive analysis challenging. Here we demonstrate an analytical method designed to target one region of the metabolome, that is, oxysterols. Since the discovery of their biological activity as ligands to nuclear receptors there has been a reawakening of interest in oxysterols and their analysis. In addition, the oxysterols, 24S- and 27-hydroxycholesterol, are currently under investigation as potential biomarkers associated with neurodegenerative disorders such as Alzheimer's disease and multiple sclerosis; widespread analysis of these lipids in clinical studies will require the development of robust, sensitive and rapid analytical techniques. In this communication we present results of an investigation of the oxysterols content of human plasma using a newly developed high-performance liquid chromatography-mass spectrometry (HPLC-MS) method incorporating charge-tagging and high-resolution MS. The method has allowed the identification in plasma of monohydroxylated cholesterol molecules, 7alpha-, 24S-, and 27-hydroxycholesterol; the cholestenetriol 7alpha,27-dihydroxycholesterol; and 3beta-hydroxycholest-5-en-27-oic acid and its metabolite 3beta,7alpha-dihydroxycholest-5-en-27-oic acid. The methodology described is also applicable for the analysis of other sterols in plasma, that is, cholesterol, 7-dehydrocholesterol, and desmosterol, as well as cholesterol 5,6- seco-sterols and steroid hormones. Although involving derivatization, sample preparation is straightforward and chromatographic analysis rapid (17 min), while the MS method offers high sensitivity (ng/mL of sterol in plasma, or pg on-column) and specificity. The methodology is suitable for targeted metabolomic analysis of sterols, oxysterols, and steroid hormones opening a "window" to view this region of the metabolome.
    Journal of Proteome Research 08/2008; 7(8):3602-12. DOI:10.1021/pr8001639 · 5.00 Impact Factor
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    ABSTRACT: Smith-Lemli-Opitz syndrome (SLOS), a severe disorder of cholesterol synthesis, is classically diagnosed prenatally by GC-MS analysis of sterols in amniotic fluid. Considering the current trend toward tandem mass spectrometry (MS/MS) methodologies, we developed prototype LC-MS/MS methods for accurate diagnosis of the disorder. 3beta-Hydroxysterols in amniotic fluid are oxidized with cholesterol oxidase to their corresponding 3-ketones, which are then derivatized with Girard P (GP) hydrazine in a "one-pot" reaction. The resulting GP-hydrazones give an improved response in electrospray (ES)-MS/MS owing to the presence of a charged quaternary nitrogen and are analyzed by reversed-phase LC-ES-MS/MS. Both capillary and conventional LC-MS/MS formats are suitable, and the method is also applicable to paper-absorbed blood spots. In a double-blind analysis of 18 amniotic fluid samples comprising 6 SLOS and 12 controls, the ratio of 7 + 8-dehydrocholesterol (7 + 8-DHC) to cholesterol was <0.02 [range 0.00-0.02, mean (SD) 0.01 (0.007)] in all control samples (intraassay variation 5.91%) and >0.20 [0.20-1.13, 0.79 (0.35)] in SLOS (intraassay variation 4.56%), corresponding to a difference in ratios between the 2 groups of at least a factor of 10. The limit of quantification was equivalent to that of 2 nL amniotic fluid injected on-column. We describe a proof-of-concept for the prenatal diagnosis of SLOS. Further developments will be necessary to automate sample handling and reduce chromatographic time for the methodology to be used in pre- and postnatal diagnosis.
    Clinical Chemistry 06/2008; 54(8):1317-24. DOI:10.1373/clinchem.2007.100644 · 7.77 Impact Factor
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    ABSTRACT: In humans, the brain accounts for about 20% of the body's free cholesterol, most of which is synthesized de novo in brain. To maintain cholesterol balance throughout life, cholesterol becomes metabolized to 24S-hydroxycholesterol, principally in neurons. In mouse, rat, and probably human, metabolism to 24S-hydroxycholesterol accounts for about 50% of cholesterol turnover; however, the route by which the remainder is turned over has yet to be elucidated. Here, we describe a novel liquid chromatography (LC) multi-stage fragmentation mass spectrometry (MS(n)) methodology for the identification, with high sensitivity (low pg), of cholesterol metabolites in rat brain. The methodology includes derivatization to enhance ionization, exact mass analysis at high resolution to identify potential metabolites, and LC-MS(n) (n=3) to allow their characterization. 24S-hydroxycholesterol was confirmed as a major oxysterol in rat brain, and other oxysterols identified for the first time in brain included 24,25-, 24,27-, 25,27-, 6,24,- 7alpha,25-, and 7alpha,27-dihydroxycholesterols. In addition, 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al and its aldol, two molecules linked to amyloidogenesis of proteins, were characterized in rat brain.
    The Journal of Lipid Research 05/2007; 48(4):976-87. DOI:10.1194/jlr.M600497-JLR200 · 4.73 Impact Factor
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    ABSTRACT: Following the sequencing of the human and other genomes, much research effort is now invested in post- genomic science, particularly in the related disciplines of proteomics and metabolomics. In this paper, we will attempt to provide an overview of mass spectrometry-based metabolomic strategies, discuss the evolution of metabolomics from its predecessor, Hmetabolite profiling", and provide some pointers to future methodological and technological direction. Current data from the authors' laboratory will also be presented, highlighting our efforts in the field of "targeted metabolomics", namely, "steroidomics in the brain".
    European Journal of Mass Spectrometry 02/2007; 13(1):45-50. DOI:10.1255/ejms.850 · 1.17 Impact Factor
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    ABSTRACT: Repeated exposure to drugs of abuse causes time-dependent neuroadaptive changes in the mesocorticolimbic system of the brain that are considered to underlie the expression of major behavioral characteristics of drug addiction. We used a 2-D gel-based proteomics approach to examine morphine-induced temporal changes in protein expression and/or PTM in the nucleus accumbens (NAc) of morphine-sensitized rats. Rats were pretreated with saline [1 mL/kg subcutaneously (s.c.)] or morphine (10 mg/kg, s.c.) once daily for 14 days and the animals were decapitated 1 day later. The NAc was extracted and proteins resolved by 2-DE. Several protein functional groups were found to be regulated in the morphine-treated group, representing cytoskeletal proteins, proteins involved in neurotransmission, enzymes involved in energy metabolism and protein degradation, and a protein that regulates translation.
    PROTEOMICS 03/2006; 6(6):2003-8. DOI:10.1002/pmic.200500045 · 3.97 Impact Factor
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    ABSTRACT: Neutral steroids have traditionally been analyzed by gas chromatography/mass spectrometry (GC/MS) after necessary derivatization reactions. However, GC/MS is unsuitable for the analysis of many conjugated steroids and those with unsuspected functional groups. Here we describe an alternative analytical method specifically designed for the analysis of oxosteroids and those with a 3beta-hydroxy-delta5 or 5alpha-hydrogen-3beta-hydroxy structure. Steroids were derivatized with Girard P (GP) hydrazine to give GP hydrazones, which are charged species and readily analyzed by matrix-assisted laser desorption/ionization mass spectrometry. The resulting [M]+ ions were then subjected to high-energy collision-induced dissociation on a tandem time-of-flight instrument. The product ion spectra give structurally informative fragment ion patterns. The sensitivity of the analytical method is such that steroid structures can be determined from low-picogram (low-femtomole) amounts of sample. The utility of the method has been demonstrated by the analysis of oxysterols extracted from rat brain.
    Analytical Chemistry 02/2006; 78(1):164-73. DOI:10.1021/ac051461b · 5.83 Impact Factor
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    ABSTRACT: Organelle proteomics is the method of choice for global analysis of cellular proteins. However, it is difficult to isolate organelles to homogeneity. Recently, correlation-profiling has been used to filter off the contaminants ad hoc and to disclose the genuine organelle-specific proteins. In the present study, we further extend the method to include subcellular compartments that contain proteins shared by multiple distinct subcellular domains. We performed correlation profiling of proteins contained in synaptic membrane and postsynaptic density (PSD) fractions isolated from rat brain. Proteins were labeled with isotope-coded affinity-tag reagents, digested with trypsin, and resulting peptides were resolved by cation exchange chromatography followed by reversed phase chromatography. Peptides were then subjected to mass spectrometry for quantification and identification. We confirm that the core PSD proteins were enriched in the PSD preparation. Other functional protein groups such as cytoskeleton-associated proteins, protein kinases and phosphatases, signaling components and regulators, as well as proteins involved in energy production partitioned to multiple organelles. When analyzed as groups, they were shown to accumulate to a lesser extent. Mitochondrial proteins and transporters were generally strongly depleted from the PSD fraction confirming that they were contaminants of the PSD preparation. Finally, immunoelectron microscopy was performed on selected proteins to validate the proteomics results, and confirm that synaptophysin that was highly depleted in the PSD preparation is localized in the presynaptic compartment, whereas LASP-1 that was slightly enriched in the PSD preparation is present in the PSD as well as other subdomains within the synapse.
    Journal of Proteome Research 06/2005; 4(3):725-33. DOI:10.1021/pr049802+ · 5.00 Impact Factor
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    ABSTRACT: Using proteomics, we investigated the temporal expression profiles of proteins in rat sciatic nerve after experimental crush. Extracts of sciatic nerves collected at 5, 10, and 35 days after injury were analyzed by two-dimensional gel electrophoresis and quantitative image analysis. Of the approximately 1,500 protein spots resolved on each gel, 121 showed significant regulation during at least one time point. Using cluster analysis, these proteins were grouped into two expression profiles of down-regulation and four of up-regulation. These profiles mainly reflected differences in cellular origins in addition to different functional roles. Mass spectrometric analysis identified 82 proteins pertaining to several functional classes, i.e. acute-phase proteins, antioxidant proteins, and proteins involved in protein synthesis/maturation/degradation, cytoskeletal (re)organization, and in lipid metabolism. Several proteins not previously implicated in nerve regeneration were identified, e.g. translationally controlled tumor protein, annexin A9/31, vitamin D-binding protein, alpha-crystallin B, alpha-synuclein, dimethylargininases, and reticulocalbin. Real-time PCR analysis of selected genes showed which were expressed in the nerve versus the dorsal root ganglion neurons. In conclusion, this study highlights the complexity and temporal aspect of the molecular process underlying nerve regeneration and points to the importance of glial and inflammatory determinants.
    Molecular &amp Cellular Proteomics 03/2005; 4(2):120-32. DOI:10.1074/mcp.M400076-MCP200 · 7.25 Impact Factor
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    ABSTRACT: The postsynaptic density contains multiple protein complexes that together relay the presynaptic neurotransmitter input to the activation of the postsynaptic neuron. In the present study we took two independent proteome approaches for the characterization of the protein complement of the postsynaptic density, namely 1) two-dimensional gel electrophoresis separation of proteins in conjunction with mass spectrometry to identify the tryptic peptides of the protein spots and 2) isolation of the trypsin-digested sample that was labeled with isotope-coded affinity tag, followed by liquid chromatography-tandem mass spectrometry for the partial separation and identification of the peptides, respectively. Functional grouping of the identified proteins indicates that the postsynaptic density is a structurally and functionally complex organelle that may be involved in a broad range of synaptic activities. These proteins include the receptors and ion channels for glutamate neurotransmission, proteins for maintenance and modulation of synaptic architecture, sorting and trafficking of membrane proteins, generation of anaerobic energy, scaffolding and signaling, local protein synthesis, and correct protein folding and breakdown of synaptic proteins. Together, these results imply that the postsynaptic density may have the ability to function (semi-) autonomously and may direct various cellular functions in order to integrate synaptic physiology.
    Journal of Biological Chemistry 02/2004; 279(2):987-1002. DOI:10.1074/jbc.M303116200 · 4.60 Impact Factor
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    ABSTRACT: To reveal the peptide contents of the visually nonidentifiable neurons from a neuronal circuit of interest, we combined retrograde labeling of neurons with mass spectrometric single cell analysis. We used the neuronal circuit involved in the copulation behavior of a freshwater snail, Lymnaea stagnalis, as a model. Central neurons that control this behavior are known to send their axons to the penis nerve and innervate the penis complex. By retrograde filling from the penis nerve with nickel-lysine, these neurons were selectively labeled darkish blue. Matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometric analyses of single stained neurons in the parietal ganglion from different animals reveal consistently the presence of several molecular ion species in the range of 800-1200 Da. From a single neuron, six molecular ion species were further characterized with MALDI time-of-flight/time-of-flight mass spectrometry, which demonstrates that the peptides are derived from a previously reported -FLRFamide precursor.
    Analytical Chemistry 08/2003; 75(13):2996-3000. DOI:10.1021/ac034057q · 5.83 Impact Factor