Comparison of Protein Expression Ratios Observed by Sixplex and Duplex TMT Labeling Method

Stable isotope labeling via isobaric derivatization of peptides is a universally applicable approach that enables concurrent identification and quantification of proteins in different samples using tandem mass spectrometry. In this study, we evaluated the performance of amine-reactive isobaric tandem mass tag (TMT), available as duplex and sixplex sets, with regard to their ability to elucidate protein expression changes. Using rat brain tissue from two different developmental time points, postnatal day 1 (p1) and 45 (p45), as a model system, we compared the protein expression ratios (p45/p1) observed using duplex TMT tags in triplicate measurements versus sixplex tag in a single LC–MS/MS analysis. A correlation of 0.79 in relative protein abundance was observed in the proteins quantified by these two sets of reagents. However, more proteins passed the criteria for significant fold change (−1.0 ≤ log₂ ratio (p45/p1) ≥ +1.0 and <i>p</i> < 0.05) in the sixplex analysis. Nevertheless, in both methods most proteins showing significant fold change were identified by multiple spectra, increasing their quantification precision. Additionally, the fold change in p45 rats against p1, observed in TMT experiments, was corroborated by a metabolic labeling strategy where relative quantification of differentially expressed proteins was obtained using ¹⁵N-labeled p45 rats as an internal standard

Obchod s uhlíkem jako možnost financování soukromých rezervací - případová studie z Reserva Silvestre Greenfields v Nikaragui

The objective of this work was to measure the amount of carbon fixed in the tree biomass in the forest of Reserva Silvestre Greenfields, on the basis of inventarization of the forest and suggest a participation of the area in various projects engaged in the support of carbon sequestration in the forest ecosystems. The forest inventarization was conducted and the following research determined the average wood density of the tree species occuring on territory, total biomass and the amount of carbon in the wood mass of the woodland. Based on the data were then suggested the most suitable organizations supporting carbon management in the forest ecosystems

DMF and MEF Induce Different Effects on Extracellular GSH.

<p>Primary cultures of human astrocytes were incubated with 1 (A) or 3 (B) μg/mL DMF, MEF or DMSO as a control. Media was collected from treated cells after 0.0, 0.5, 1.0, 6, 12 or 24 hours of treatment, and total extracellular glutathione was measured as relative luminescence units (RLU). Each point represents the mean of triplicate determinations (± SD). Dotted line represents average basal RLU levels. *, <i>p</i><0.01 for DMF versus MEF at indicated time point. <i>P</i> values are based on two-way ANOVA with Tukey’s post-test for multiple comparisons.</p

DMF and MEF Differentially Modify KEAP1 Cysteine Residues.

<p>KEAP1 transfected HEK 293FT cells were treated with DMF and MEF salts (Ca²⁺, Mg²⁺, Zn²⁺) at 3 μg/mL (A, C) or 6 μg/mL (B, D). Resulting cysteine modifications on KEAP1 were analyzed using mass spectrometry. Percent modification of KEAP1 cysteine residues with DMF or MEF was determined relative to DMSO control treated cells. (A, B) Representation of percent cysteine modification of analyzed KEAP1 cysteine residues in the presence of 3 (A) or 6 (B) μg/mL DMF or MEF. Each bar represents the means of quadruplicate determinations (± SD). (C, D) Box-whisker plots demonstrate the means, quartiles, and max-min of KEAP1 cysteine residues modified by greater than 10 percent in A and B. *, <i>p</i><0.05. ***, <i>p</i><0.001. ****, <i>p</i><0.0001. <i>P</i> values are based on two-way analysis of variance (ANOVA) with Sidak’s post-test for multiple comparisons.</p

Structure and basic properties of DMF, MEF salts and Fumaric Acid.

<p>DMF and MEF are esters of fumaric acid, which is not pharmacologically active. The active moiety(ies) of DMF and MEF confer their chemical and physical properties.</p

DMF and MEF Induce Different Effects on Cellular GSH.

<p>Primary cultures of human astrocytes were incubated with 1 (A) or 3 (B) μg/mL DMF, MEF, or DMSO as a control. Treated cells were harvested after 0.0, 0.5, 1.0, 6, 12, and 24 hours of treatment, and total cellular GSH was measured as relative luminescence units (RLU). Each point represents the mean of triplicate determinations (± SD). Dotted line represents average basal RLU levels. *, <i>p</i><0.01 for DMF versus MEF at indicated time point. <i>P</i> values are based on two-way ANOVA with Tukey’s post-test for multiple comparisons.</p