19 research outputs found
Vacuum Ultraviolet Photodissociation and Fourier Transform–Ion Cyclotron Resonance (FT-ICR) Mass Spectrometry: Revisited
We revisited the implementation of
193 nm ultraviolet photodissociation
(UVPD) within the ion cyclotron resonance (ICR) cell of a Fourier
transform–ion cyclotron resonance (FT-ICR) mass spectrometer.
UVPD performance characteristics were examined in the context of recent
developments in the understanding of UVPD and in-cell tandem mass
spectrometry. Efficient UVPD and photo-ECD of a model peptide and
proteins within the ICR cell of a FT-ICR mass spectrometer are accomplished
through appropriate modulation of laser pulse timing, relative to
ion magnetron motion and the potential applied to an ion optical element
upon which photons impinge. It is shown that UVPD yields efficient
and extensive fragmentation, resulting in excellent sequence coverage
for model peptide and protein cations
Concentration-Response of <i>pTpfcp/Sil3-CRY:fcp/nat-</i>transformed Diatoms Exposed to Ribose.
<p>Normalized ΔFRET values (ΔF<sub>n</sub>) for individual cells treated with ribose. The EC<sub>50</sub> of 23.3±4.7 mM (mean ± S.E.) corresponds to the ribose concentration resulting in 50% of the maximal ΔF<sub>n</sub> value.</p
Ribose-Dependent FRET Imaging in Living, Transformed Diatoms and Isolated Biosilica.
<p>(A) Time lapse imaging of living cells was used to image the CyPet and YPet fluorescence before and after addition of 300 mM ribose. Fluorescent images were captured simultaneously in both fluorescent channels every min for 40 min. Ribose was added to the cells at 20 min following initial imaging. The FRET ratio (530/485 nm RFU ratio) decreases upon addition of ribose. (B) Similarly, time lapse imaging of biosilica cell walls was carried out for 10 min with the addition of 300 mM ribose after 5 min. The FRET ratio decreases in response to ribose addition.</p
Mass Spectrometry for Amino Acid Sequence Verification of the Deduced CRY Protein.
<p>Protein samples were digested with gluC and trypsin proteases separately and analyzed using high resolution LC MS/MS. Mass spectrometry verified 97% of the deduced recombinant CRY sequence. The remaining 3% was either unidentified or represented 1 amino acid mismatch (*). Blue shading = CyPet sequence; Red shading = RBP sequence; Yellow shading = YPet sequence; Gray = linker sequences; Black Lines = His<sub>6</sub> and Xpress™ tags.</p
Fluorescent Images of Transformed and Untransformed Living Diatoms and Isolated Biosilica.
<p>(A) Fluorescence microscopy: Live transformed cells and isolated biosilica frustules were imaged for brightfield to display cell structure and for cyan fluorescence of CyPet and red auto-fluorescence to show their respective localization in the biosilica and chloroplasts. The lack of red auto-fluorescence in the isolated biosilica highlights the absence of chloroplasts and therefore, the lack of cellular material within the biosilica cell wall. (B) Imaging flow cytometry: <i>pTpfcp/Sil3-CRY:fcp/nat-</i> transformed (TR) and untransformed (WT) cells were imaged for brightfield, cyan fluorescence of CyPet, yellow fluorescence of YPet from FRET, and chloroplast auto-fluorescence. Cyan, yellow, and red images were merged to highlight the fluorescence of CyPet and YPet flanking the chloroplasts. Only the red auto-fluorescence of chloroplasts was detected in the WT cells.</p
Model for Ribose-Induced Conformational Change in CRY and Associated Decrease in FRET.
<p>Unbound: In the absence of ribose, the ribose-binding protein (RBP) is in an open configuration, with C and Y, which are attached to the amino and carboxyl termini of RBP, respectively, in close proximity to each other. When CRY is excited at 435 nm, the emission from C excites Y, resulting in a high 530/485 emission intensity ratio. Bound: Binding of ribose (R) by RBP induces a conformational change in RBP that separates the amino and carboxyl termini of RBP, thereby increasing the distance between C and Y. This increase in the distance decreases the energy transfer between the two fluorescent proteins, thereby decreasing the 530/485 emission intensity ratio. Thus, increases in ribose concentrations result in decreases in FRET.</p
Advanced Solvent Based Methods for Molecular Characterization of Soil Organic Matter by High-Resolution Mass Spectrometry
Soil organic matter (SOM), a complex,
heterogeneous mixture of
above and belowground plant litter and animal and microbial residues
at various degrees of decomposition, is a key reservoir for carbon
(C) and nutrient biogeochemical cycling in soil based ecosystems.
A limited understanding of the molecular composition of SOM limits
the ability to routinely decipher chemical processes within soil and
accurately predict how terrestrial carbon fluxes will respond to changing
climatic conditions and land use. To elucidate the molecular-level
structure of SOM, we selectively extracted a broad range of intact
SOM compounds by a combination of different organic solvents from
soils with a wide range of C content. Our use of electrospray ionization
(ESI) coupled with Fourier transform ion cyclotron resonance mass
spectrometry (FTICR MS) and a suite of solvents with varying polarity
significantly expands the inventory of the types of organic molecules
present in soils. Specifically, we found that hexane is selective
for lipid-like compounds with very low O/C ratios (<0.1); water
(H<sub>2</sub>O) was selective for carbohydrates with high O/C ratios;
acetonitrile (ACN) preferentially extracts lignin, condensed structures,
and tannin polyphenolic compounds with O/C > 0.5; methanol (MeOH)
has higher selectivity toward compounds characterized with low O/C
< 0.5; and hexane, MeOH, ACN, and H<sub>2</sub>O solvents increase
the number and types of organic molecules extracted from soil for
a broader range of chemically diverse soil types. Our study of SOM
molecules by ESI FTICR MS revealed new insight into the molecular-level
complexity of organics contained in soils. We present the first comparative
study of the molecular composition of SOM from different ecosystems
using ultra high-resolution mass spectrometry
Formularity: Software for Automated Formula Assignment of Natural and Other Organic Matter from Ultrahigh-Resolution Mass Spectra
Ultrahigh
resolution mass spectrometry, such as Fourier transform
ion cyclotron resonance mass spectrometry (FT ICR MS), can resolve
thousands of molecular ions in complex organic matrices. A Compound
Identification Algorithm (CIA) was previously developed for automated
elemental formula assignment for natural organic matter (NOM). In
this work, we describe software Formularity with a user-friendly interface
for CIA function and newly developed search function Isotopic Pattern
Algorithm (IPA). While CIA assigns elemental formulas for compounds
containing C, H, O, N, S, and P, IPA is capable of assigning formulas
for compounds containing other elements. We used halogenated organic
compounds (HOC), a chemical class that is ubiquitous in nature as
well as anthropogenic systems, as an example to demonstrate the capability
of Formularity with IPA. A HOC standard mix was used to evaluate the
identification confidence of IPA. Tap water and HOC spike in Suwannee
River NOM were used to assess HOC identification in complex environmental
samples. Strategies for reconciliation of CIA and IPA assignments
were discussed. Software and sample databases with documentation are
freely available
Phylogenetic bins and statistics.
a<p>: Based on blastn coverage of reference genome, rather than phylogenetic markers. <sup>b</sup>: Including additional cluster at 12.0 read coverage that likely corresponds to a Sphaerobacter megaplasmid. <sup>c</sup>: See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068465#pone.0068465.s003" target="_blank">File S3</a>.</p
Indexing Permafrost Soil Organic Matter Degradation Using High-Resolution Mass Spectrometry
<div><p>Microbial degradation of soil organic matter (SOM) is a key process for terrestrial carbon cycling, although the molecular details of these transformations remain unclear. This study reports the application of ultrahigh resolution mass spectrometry to profile the molecular composition of SOM and its degradation during a simulated warming experiment. A soil sample, collected near Barrow, Alaska, USA, was subjected to a 40-day incubation under anoxic conditions and analyzed before and after the incubation to determine changes of SOM composition. A CHO index based on molecular C, H, and O data was utilized to codify SOM components according to their observed degradation potentials. Compounds with a CHO index score between –1 and 0 in a water-soluble fraction (WSF) demonstrated high degradation potential, with a highest shift of CHO index occurred in the N-containing group of compounds, while similar stoichiometries in a base-soluble fraction (BSF) did not. Additionally, compared with the classical H:C vs O:C van Krevelen diagram, CHO index allowed for direct visualization of the distribution of heteroatoms such as N in the identified SOM compounds. We demonstrate that CHO index is useful not only in characterizing arctic SOM at the molecular level but also enabling quantitative description of SOM degradation, thereby facilitating incorporation of the high resolution MS datasets to future mechanistic models of SOM degradation and prediction of greenhouse gas emissions.</p></div