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_n) for individual cells treated with ribose. The EC₅₀ of 23.3±4.7 mM (mean ± S.E.) corresponds to the ribose concentration resulting in 50% of the maximal ΔF_n 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 = His₆ 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 (H₂O) 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 H₂O 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. ^b: Including additional cluster at 12.0 read coverage that likely corresponds to a Sphaerobacter megaplasmid. ^c: 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

Normalized protein abundance within each of the three proteome fractions, by phylogenetic bins.

<p>Normalized protein abundance within each of the three proteome fractions, by phylogenetic bins.</p