101 research outputs found
Activated Ion-Electron Transfer Dissociation Enables Comprehensive Top-Down Protein Fragmentation
Here
we report the first demonstration of near-complete sequence
coverage of intact proteins using activated ion-electron transfer
dissociation (AI-ETD), a method that leverages concurrent infrared
photoactivation to enhance electron-driven dissociation. AI-ETD produces
mainly c/z-type product ions and provides comprehensive (77–97%)
protein sequence coverage, outperforming HCD, ETD, and EThcD for all
proteins investigated. AI-ETD also maintains this performance across
precursor ion charge states, mitigating charge-state dependence that
limits traditional approaches
Assessment of the proteomic data.
<p>(A) Venn diagram of spore proteins and yeast proteins identified. 2232 and 2192 proteins were identified from spores and yeast, respectively, with a majority of 1858 existing in both cell types. (B) Virtual two-dimensional gel diagrams of the predicted <i>C</i>. <i>neoformans</i> proteome (upper panel) and identified proteins in either yeast (middle panel) or spores (lower panel). Each dot represents a protein, with the x-axis showing isoelectric point (pI) and the y-axis as molecular weight (MW, Dalton).</p
Protein Composition of Infectious Spores Reveals Novel Sexual Development and Germination Factors in <i>Cryptococcus</i>
<div><p>Spores are an essential cell type required for long-term survival across diverse organisms in the tree of life and are a hallmark of fungal reproduction, persistence, and dispersal. Among human fungal pathogens, spores are presumed infectious particles, but relatively little is known about this robust cell type. Here we used the meningitis-causing fungus <i>Cryptococcus neoformans</i> to determine the roles of spore-resident proteins in spore biology. Using highly sensitive nanoscale liquid chromatography/mass spectrometry, we compared the proteomes of spores and vegetative cells (yeast) and identified eighteen proteins specifically enriched in spores. The genes encoding these proteins were deleted, and the resulting strains were evaluated for discernable phenotypes. We hypothesized that spore-enriched proteins would be preferentially involved in spore-specific processes such as dormancy, stress resistance, and germination. Surprisingly, however, the majority of the mutants harbored defects in sexual development, the process by which spores are formed. One mutant in the cohort was defective in the spore-specific process of germination, showing a delay specifically in the initiation of vegetative growth. Thus, by using this in-depth proteomics approach as a screening tool for cell type-specific proteins and combining it with molecular genetics, we successfully identified the first germination factor in <i>C</i>. <i>neoformans</i>. We also identified numerous proteins with previously unknown functions in both sexual development and spore composition. Our findings provide the first insights into the basic protein components of infectious spores and reveal unexpected molecular connections between infectious particle production and spore composition in a pathogenic eukaryote.</p></div
Organic Acid Quantitation by NeuCode Methylamidation
We have developed a multiplexed quantitative
analysis method for
carboxylic acids by liquid chromatography high resolution mass spectrometry.
The method employs neutron encoded (NeuCode) methylamine labels (<sup>13</sup>C or <sup>15</sup>N enriched) that are affixed to carboxylic
acid functional groups to enable duplex quantitation via mass defect
measurement. This work presents the first application of NeuCode quantitation
to small molecules. We have applied this technique to detect adulteration
of olive oil by quantitative analysis of fatty acid methyl amide derivatives,
and the quantitative accuracy of the NeuCode analysis was validated
by GC/MS. Currently, the method enables duplex quantitation and is
expandable to at least 6-plex analysis
<i>isp2Δ</i> spores show a delay in germination.
<p>(A) <i>isp2Δ</i> spores have a germination defect on solid YPD medium. Colonies of wild type and <i>isp2Δ</i> strains after growth at room temperature for 63h (germination) and 51h (vegetative growth). Scale bars, 100μm (5x magnification). (B) Quantification of colony sizes using ImageJ. The average colony size of <i>isp2Δ</i> spores was only 20.5% of wild type spores, whereas the average colony size of <i>isp2Δ</i> yeast was 45.7% of wild type yeast. The difference in size between colonies from yeast growth and spore germination for <i>isp2Δ</i> strains was significant (p = 7.1x10<sup>-48</sup>) but not for the wild type strain (p = 7.8x10<sup>-1</sup>). Data represent number (n) of independent experiments and are shown as a mean ± SD. An unpaired two-sided Student's t-test was used to assess significance. (C) Germination delay for <i>isp2Δ</i> spores in liquid YPD media. Optical density at a wavelength of 600nm (OD<sub>600</sub>) was measured every 3min over 50h. The y-axis shows OD<sub>600</sub> and the x-axis shows time in hours (h). Plots are representative of three independent experiments. (D) Average time taken to double initial OD<sub>600</sub>. Quantified doubling times were nearly identical for wild type and <i>isp2Δ</i> yeast (p = 0.38); however, <i>isp2Δ</i> spores took significantly longer than wild type to double the population (p = 1.2×10<sup>−10</sup>). Data represent number (n) of independent experiments and are shown as mean ± SD. An unpaired two-sided Student's t-test was used to assess significance. (E) Morphological changes during germination of <i>isp2Δ</i> and wild type spores. Spores were exposed to YPD liquid media to trigger germination at room temperature and photographed at 0h and 12h. Scale bars, 5μm (1000× magnification).</p
Full-Featured Search Algorithm for Negative Electron-Transfer Dissociation
Negative electron-transfer
dissociation (NETD) has emerged as a
premier tool for peptide anion analysis, offering access to acidic
post-translational modifications and regions of the proteome that
are intractable with traditional positive-mode approaches. Whole-proteome
scale characterization is now possible with NETD, but proper informatic
tools are needed to capitalize on advances in instrumentation. Currently
only one database search algorithm (OMSSA) can process NETD data.
Here we implement NETD search capabilities into the Byonic platform
to improve the sensitivity of negative-mode data analyses, and we
benchmark these improvements using 90 min LC–MS/MS analyses
of tryptic peptides from human embryonic stem cells. With this new
algorithm for searching NETD data, we improved the number of successfully
identified spectra by as much as 80% and identified 8665 unique peptides,
24 639 peptide spectral matches, and 1338 proteins in activated-ion
NETD analyses, more than doubling identifications from previous negative-mode
characterizations of the human proteome. Furthermore, we reanalyzed
our recently published large-scale, multienzyme negative-mode yeast
proteome data, improving peptide and peptide spectral match identifications
and considerably increasing protein sequence coverage. In all, we
show that new informatics tools, in combination with recent advances
in data acquisition, can significantly improve proteome characterization
in negative-mode approaches
Spores derived from wild type by <i>isp2Δ</i> crosses show wild type rates of germination.
<p>(A) Spores purified from WT × <i>isp2Δ</i> strains were grown in YPD and measured via optical density (OD<sub>600</sub>) every 3min over 50h. The y-axis shows OD<sub>600</sub> and the x-axis shows time in hours (h). Plots are representative of three independent experiments. (B) Model of Isp2 activity during germination. Germination encompasses two stages: a morphological transition and growth initiation before the active replication of vegetative growth. Isp2 protein (black triangles) is present in mature spores from WT × WT crosses and persists during germination through the morphological transition to contribute to optimal growth initiation. In contrast, there is no Isp2 in spores from <i>isp2Δ</i> × <i>isp2Δ</i> crosses, and thus, a delay of ~2h during germination occurs, specifically during the growth initiation phase. Notably, spores from WT × <i>isp2Δ</i> crosses do not show a delay in germination and therefore contain Isp2 protein similar to wild type spores, regardless of genotype. Spores are shown as ovals with stalks, whereas yeast are shown as spheres. Large and small spheres together represent budding yeast. Isp2 protein is represented by black triangles.</p
Development of a GC/Quadrupole-Orbitrap Mass Spectrometer, Part II: New Approaches for Discovery Metabolomics
Identification
of unknown peaks in gas chromatography/mass spectrometry
(GC/MS)-based discovery metabolomics is challenging, and remains necessary
to permit discovery of novel or unexpected metabolites that may elucidate
disease processes and/or further our understanding of how genotypes
relate to phenotypes. Here, we introduce two new technologies and
an analytical workflow that can facilitate the identification of unknown
peaks. First, we report on a GC/Quadrupole-Orbitrap mass spectrometer
that provides high mass accuracy, high resolution, and high sensitivity
analyte detection. Second, with an “intelligent” data-dependent
algorithm, termed molecular-ion directed acquisition (MIDA), we maximize
the information content generated from unsupervised tandem MS (MS/MS)
and selected ion monitoring (SIM) by directing the MS to target the
ions of greatest information content, that is, the most-intact ionic
species. We combine these technologies with <sup>13</sup>C- and <sup>15</sup>N-metabolic labeling, multiple derivatization and ionization
types, and heuristic filtering of candidate elemental compositions
to achieve (1) MS/MS spectra of nearly all intact ion species for
structural elucidation, (2) knowledge of carbon and nitrogen atom
content for every ion in MS and MS/MS spectra, (3) relative quantification
between alternatively labeled samples, and (4) unambiguous annotation
of elemental composition
Eighteen genes encoding spore-enriched proteins.
<p><sup>a</sup>. Genes encoding proteins with no obvious homologs were named <i>ISP</i> for <u>I</u>dentified <u>S</u>pore <u>P</u>rotein.</p><p>Eighteen genes encoding spore-enriched proteins.</p
Characterization of the sexual development of deletion mutants for spore-enriched proteins.
<p>(A) <i>rsc9Δ</i> strains show defects in mating, while <i>isp1Δ</i> and <i>bch1Δ</i> strains do not (top panels). Strains were mixed, incubated on V8 plates for 24h at 25°C, scraped up and visualized. Fusants (indicated by arrows) and yeast were counted. The number of fusants as a portion of total cell number is represented graphically for each strain (bottom panel). Data represent four individual experiments and are shown as mean ± standard deviation (SD). Scale bars, 10μm (100x magnification). (B) <i>rsc9Δ</i>, <i>isp1Δ</i>, and <i>bch1Δ</i> crosses showed much less robust filamentation 24h after the start of sexual development. Panels show the periphery of a spot of a cross on V8 plate for 24h at 25°C. Scale bars, 50μm (200x magnification). (C) <i>ddi1Δ</i>, <i>dst1Δ</i>, and <i>top1Δ</i> strains showed defects in spore formation. Both wild type and mutant crosses showed robust filamentation after 5 days on V8 plate (upper panels), but only wild type produced chains of spores (lower panels; arrows indicate basidia and triangles indicate spore chains). Mutants produced basidia without spore chains. Scale bars, 50μm (200x magnification) for upper panels and 10μm for lower panels (400x magnification). Spore isolations using density gradient centrifugation yielded 1%±1%, 2%±1%, and 0%±0% spores relative to wild type crosses from <i>ddi1Δ</i>, <i>dst1Δ</i>, and <i>top1Δ</i> crosses, respectively. (D) Quantified spore yield from density gradient purifications of mutant strains relative to wild type strains. <i>emc3Δ</i> and <i>gre202Δ</i> crosses yielded reproducible decreases in spore yield (approx. 2–4 fold) relative to wild type strains, whereas <i>isp2Δ</i> crosses produced more spores (approx. 1.5-fold higher) relative to wild type strains. Data represent number (n) of independent experiments and are shown as mean ± SD.</p
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