The N-methyl-d-aspartate-type glutamate receptor (NMDAR)-associated multiprotein complexes are in... more The N-methyl-d-aspartate-type glutamate receptor (NMDAR)-associated multiprotein complexes are indispensable for synaptic plasticity and cognitive functions. While purification and proteomic analyses of these signaling complexes have been performed in adult rodent and human brain, much less is known about the protein composition of NMDAR complexes in the developing brain and their modifications by neonatal hypoxic-ischemic (HI) brain injury. In this study, the postsynaptic density proteins were prepared from postnatal day 9 naïve, sham-operated and HI-injured mouse cortex. The GluN2B-containing NMDAR complexes were purified by immunoprecipitation with a mouse GluN2B antibody and subjected to mass spectrometry analysis for determination of the GluN2B binding partners. A total of 71 proteins of different functional categories were identified from the naïve animals as native GluN2B-interacting partners in the developing mouse brain. Neonatal HI reshaped the postsynaptic GluN2B interact...
Proteomics of the synapses and postsynaptic densities (PSDs) have provided a deep understanding o... more Proteomics of the synapses and postsynaptic densities (PSDs) have provided a deep understanding of protein composition and signal networks in the adult brain, which underlie neuronal plasticity and neurodegenerative or psychiatric disorders. However, there is a paucity of knowledge about the architecture and organization of PSDs in the immature brain, and how it is modified by brain injury in an early developing stage. Mass spectrometry (MS)-based proteomic analysis was performed on PSDs prepared from cortices of postnatal day 9 naïve mice or pups which had suffered hypoxic-ischemic (HI) brain injury. 512 proteins of different functional groups were identified from PSDs collected 1 h after HI injury, among which 60 have not been reported previously. Seven newly identified proteins involved in neural development were highlighted. HI injury increased the yield of PSDs at early time points upon reperfusion, and multiple proteins were recruited into PSDs following the insult. Quantitati...
The authors note that they "unintentionally labeled Fig. 'Grazer biomass (g DW) *shoot (cm) -1 ' ... more The authors note that they "unintentionally labeled Fig. 'Grazer biomass (g DW) *shoot (cm) -1 ' instead of 'Grazer biomass (mg DW) *shoot (cm) -1 .' " The corrected figure and its legend appear below. This error does not affect the conclusions of the article.
Quadrupolar excitation and collisional cooling for axialization and high pressure trapping of ions in Fourier transform ion cyclotron resonance mass spectrometry
International Journal of Mass Spectrometry and Ion Processes, 1992
A recently developed technique for axialization of ions in a Penning (i.e. hyperbolic) trap has b... more A recently developed technique for axialization of ions in a Penning (i.e. hyperbolic) trap has been adapted for Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry in a dual cubic trap instrument. A resonant alternating electric azimuthal quadrupolar field at the combination frequency, [omega]c = [omega]+ + [omega]-, converts ion magnetron motion into collision-damped cyclotron motion. The usual collision- induced
Determination of Ion Magnetron Radial Distribution in Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
Rapid Communications in Mass Spectrometry, 1996
The spatial distribution of the ion cyclotron orbit 'guiding centers' (magnetron ... more The spatial distribution of the ion cyclotron orbit 'guiding centers' (magnetron radii) in a Penning trap determines virtually all aspects of Fourier transform ion cyclotron resonance (FTICR) mass spectrometry performance. Here, we demonstrate experimentally a simple method for determining that distribution, based on measuring FTICR mass spectral peak height following dipolar excitation/dipolar detection at various stages of magnetron radial expansion. No instrumental modifications are needed. Such data will make it possible to evaluate theoretical models of space charge and to quantitate and optimize ion formation, ion injection, ion trapping, ion transfer, photodissociation and ultrahigh-resolution FTICR experiments.
Laser-induced Fluorescence of Ba+ Ions Trapped and Mass-selected in a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer
Rapid Communications in Mass Spectrometry, 1996
We present the design and preliminary results from a Fourier transform ion cyclotron resonance (I... more We present the design and preliminary results from a Fourier transform ion cyclotron resonance (ICR) mass spectrometer developed for the direct detection of UV/visible laser-induced fluorescence of trapped, mass-selected, gas-phase ions. A 3 T superconducting magnet and an open-ended multi-section cylindrical Penning trap capture and confine ions created by electron impact or laser desorption. Azimuthal quadrupolar excitation in the presence of ion/neutral collisions cools, axializes and mass selects ions as they fill the trap. A pulsed dye laser pumped by an Nd:YAG laser provides electronic energy excitation. A Brewster window and baffles on each side of the vacuum chamber reduce the scattered light from the excitation laser. Laser-induced fluorescence is collected from mirrors and lenses and directed through a quartz window and fiber-optic bundle to a photomultiplier. The ICR and optical events are controlled by a modular ICR data station and GPIB and RS-232 interfaces. An excitation spectrum is demonstrated for atomic Ba+ ions, and should extend to laser-induced fluorescence of virtually any stable positive or negative gas-phase ions of arbitrary molecular weight: molecular or quasimolecular ions, fragment ions, adduct ions, and ions formed from ion/molecule reactions.
Stereoscopic Views of Three-dimensional Ion Trajectories in Ion Cyclotron Resonance and Quadrupole Ion Traps
Rapid Communications in Mass Spectrometry, 1996
The trajectory of an ion in a Penning (ion cyclotron resonance) or Paul (quadrupole) ion trap is ... more The trajectory of an ion in a Penning (ion cyclotron resonance) or Paul (quadrupole) ion trap is complex, typically consisting of two or three simultaneous linear or circular motions. It is therefore useful to be able to view such trajectories three-dimensionally, to be able to separate the component motions and better understand how to trap and manipulate ions so as to measure ion mass, charge and energy, and determine ion structure and reactivity. Here, we present a simple algorithm (in Basic for a personal computer) to generate stereoscopic images for viewing trapped-ion three-dimensional trajectories. Stereoscopic projection is particularly useful for visualizing multiple motional modes, as for the three simultaneous cyclotron, magnetron and trapping motions in an ion cyclotron resonance ion trap. Examples of trajectories of ions in Penning and Paul ion traps are shown, as well as a magnetic resonance magnetization trajectory during conventional azimuthal dipolar excitation. Images are available in color video display as well as hard copy, c-code is provided.
Brassinosteroid (BR) regulates gene expression and plant development through a receptor kinasemed... more Brassinosteroid (BR) regulates gene expression and plant development through a receptor kinasemediated signal transduction pathway 1 . Despite many components of the pathway identified, how the BR signal is transduced from the cell surface to the nucleus remains unclear 2 . Here we describe a complete BR signaling pathway by elucidating the key missing steps of the pathway. We show that phosphorylation of BSK1 by the BR receptor kinase BRI1 promotes BSK1 binding to the BSU1 phosphatase, and BSU1 inactivates the GSK3-like kinase BIN2 by dephosphorylating a conserved phospho-tyrosine residue (pTyr200). Mutations that affect phosphorylation/ dephosphorylation of BIN2 pTyr200 (bin2-1, bin2-Y200F, and quadruple loss-of-function of BSU1-related phosphatases) support an essential role for BSU1-mediated BIN2 dephosphorylation in BR-dependent plant growth. These results demonstrate direct sequential BR activation of BRI1, BSK1, and BSU1, and inactivation of BIN2, leading to accumulation of unphosphorylated BZR transcription factors in the nucleus. This study establishes a fully connected BR signaling pathway and provides new insight into the mechanism of GSK3 regulation.
SummaryThe infectivity and persistence of Mycobacterium tuberculosis requires the utilization of ... more SummaryThe infectivity and persistence of Mycobacterium tuberculosis requires the utilization of host cell cholesterol. We have examined the specific role of cytochrome P450 CYP125A1 in the cholesterol degradation pathway using genetic, biochemical and high‐resolution mass spectrometric approaches. The analysis of lipid profiles from cells grown on cholesterol revealed that CYP125A1 is required to incorporate the cholesterol side‐chain carbon atoms into cellular lipids, as evidenced by an increase in the mass of the methyl‐branched phthiocerol dimycocerosates. We observed that cholesterol‐exposed cells lacking CYP125A1 accumulate cholest‐4‐en‐3‐one, suggesting that this is a physiological substrate for this enzyme. Reconstitution of enzymatic activity with spinach ferredoxin and ferredoxin reductase revealed that recombinant CYP125A1 indeed binds both cholest‐4‐en‐3‐one and cholesterol, efficiently hydroxylates both of them at C‐27, and then further oxidizes 27‐hydroxycholest‐4‐en‐3...
The emergence of efficient fragmentation methods such as electron capture dissociation (ECD) or e... more The emergence of efficient fragmentation methods such as electron capture dissociation (ECD) or electron transfer dissociation (ETD) provide the opportunity for detailed structural characterization of heavily covalently modified large peptides and small proteins such as intact histones. Even with effective gas phase ion isolation so that a single molecular precursor ion is selected, the MS/MS spectrum of a heavily modified peptide may reveal the presence of a mixture of peptides with the same amino acid sequence and the same total number of PTM moieties (same PTM composition) but having different PTM configurations or site specific occupancy isoforms. Currently available data analysis methods depend on a deisotoping procedure, which becomes less effective when spectra (fragmentation patterns) contain many overlapping isotopic distributions. Peptide database search engines can only identify the most abundant PTM configuration (PTM arrangement on different residues) in such mixtures. In order to identify all the PTM configurations present in these mixtures and to estimate their relative abundances, we have extended our fragment assignment by visual assistance (FAVA) program to search for ions representing all possible configurations, subjected to the total PTM composition constraint. This results in the identification of PTM configurations supported by unique fragment ions, and their relative abundances are estimated by use of a non-negative least square procedure.
Uploads
Papers by Shenheng Guan