Mass spectra of a portion of the heat maps of VTS-270 and Trappsol Cyclo showing the m/z region dominated by singly charged NH4+ adduct ions.

<p>The spectra represent the areas in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175478#pone.0175478.g001" target="_blank">Fig 1</a> that are labeled “Region 1.” See text for detailed description of the spectra. The numbers above the most intense ions of each spectrum show the degrees of substitution of the ammonium adduct for that species (eg, nominal mass 1384, corresponding to the ammonium adduct of a degree of substitution equal to 4). The spectra show clear differences between VTS-270 and Trappsol Cyclo in the distributions of ion intensities corresponding to the degrees of substitution, with Trappsol Cyclo having a greater range and higher degrees of substitution.</p

Ion mobility false color representations (“heat map”) of VTS-270 and Trappsol Cyclo.

<p>Ion mobility drift time (msec) is shown on the x axis and m/z on the y axis. Heat map intensities are shown with colors ranging from lowest intensity in blue to highest intensity in red. Materials were infused under the same preparation conditions and at the same concentration of HPβCD. Trappsol Cyclo exhibits greater chemical heterogeneity than VTS-270 as observed by the more complex overall heat map and the m/z singly charged trend line (long oval-shaped area), the region labeled “higher order complexes,” and the region corresponding to triply charged dimers.</p

Ions as [M₂+H + NH₄]⁺².

<p>Ions as [M₂+H + NH₄]⁺².</p

Fraction of dimers as a function of the degree of substitution.

<p>Fractional intensity of homodimer ions, relative to total hydroxypropyl-beta-cyclodextrin (HPβCD) ion signal, plotted as a function of the degree of substitution (A); fractional intensity of heterodimer ions, relative to total HPβCD ion signal, plotted as a function of the degree of substitution (B). The fractional intensity of ions as heterodimers and homodimers is greater with Trappsol Cyclo (red circles) than with VTS-270 (green triangles).</p

Mass spectra of a portion of the heat maps of VTS-270 and Trappsol Cyclo showing the m/z region dominated by triply charged dimeric ions.

<p>Ions A = [DS_n +2NH₄ +H]⁺³ and Ions B = [DS_n+DS_n-1+2NH₄+H]⁺³. See text for detailed description of the spectra. The spectra show clear differences with substantial intensity of triply charged dimers for Trappsol Cyclo but not for VTS-270.</p

Structure of 2-hydroxypropyl-β-cyclodextrin (HPβCD) where R = H or CH₂-CHOH-CH₃.

<p>There are 21 (red) hydroxyl groups on βCD which are potential substitution sites for the condensation reaction with propylene oxide to yield various species of HPβCD with different degrees of substitution depending upon how many sites are substituted.</p

Ions as [M+NH₄]⁺.

<p>Ions as [M+NH₄]⁺.</p

An Efficient Approach to Evaluate Reporter Ion Behavior from MALDI-MS/MS Data for Quantification Studies Using Isobaric Tags

Protein quantification, identification, and abundance determination are important aspects of proteome characterization and are crucial in understanding biological mechanisms and human diseases. Different strategies are available to quantify proteins using mass spectrometric detection, and most are performed at the peptide level and include both targeted and untargeted methodologies. Discovery-based or untargeted approaches oftentimes use covalent tagging strategies (i.e., iTRAQ, TMT), where reporter ion signals collected in the tandem MS experiment are used for quantification. Herein we investigate the behavior of the iTRAQ 8-plex chemistry using MALDI-TOF/TOF instrumentation. The experimental design and data analysis approach described is simple and straightforward, which allows researchers to optimize data collection and proper analysis within a laboratory. iTRAQ reporter ion signals were normalized within each spectrum to remove peptide biases. An advantage of this approach is that missing reporter ion values can be accepted for purposes of protein identification and quantification without the need for ANOVA analysis. We investigate the distribution of reporter ion peak areas in an equimolar system and a mock biological system and provide recommendations for establishing fold-change cutoff values at the peptide level for iTRAQ data sets. These data provide a unique data set available to the community for informatics training and analysis

2D-GE Image of CHM2A Protein Spot.

<p>Representative 2D-GE images of CHM2A expression over the time course study. Arrows indicate the protein spot of interest. Spots for each genotype and at each time point represent triplicate analysis of a protein pool comprised of n = 4 to 6 cerebella lysates.</p

Summary of 2D-GE Differential Protein Spots.

<p>Venn diagram depicting gel spots that were identified at each time point. Numbers noted in parenthesis are the total number spots with differential intensity identified at that specific time point. Within each time point the breakdown of spots is provided in which the gel intensity suggested either increased (up arrow) or decreased (down arrow) expression relative to the control. A total of 109 spots were differentially expressed. In week one, 22 were unique to this time point whereas 8 were unique to week three and 37 were unique to week five. From the MS analysis, a total of 77 unique proteins were identified in both the mutant and control (paired) gel spots. The protein identifications included 49 increased and 22 decreased.</p