Cross-Platform Comparison of Untargeted and Targeted Lipidomics Approaches on Aging Mouse Plasma.

Lipidomics - the global assessment of lipids - can be performed using a variety of mass spectrometry (MS)-based approaches. However, choosing the optimal approach in terms of lipid coverage, robustness and throughput can be a challenging task. Here, we compare a novel targeted quantitative lipidomics platform known as the Lipidyzer to a conventional untargeted liquid chromatography (LC)-MS approach. We find that both platforms are efficient in profiling more than 300 lipids across 11 lipid classes in mouse plasma with precision and accuracy below 20% for most lipids. While the untargeted and targeted platforms detect similar numbers of lipids, the former identifies a broader range of lipid classes and can unambiguously identify all three fatty acids in triacylglycerols (TAG). Quantitative measurements from both approaches exhibit a median correlation coefficient (r) of 0.99 using a dilution series of deuterated internal standards and 0.71 using endogenous plasma lipids in the context of aging. Application of both platforms to plasma from aging mouse reveals similar changes in total lipid levels across all major lipid classes and in specific lipid species. Interestingly, TAG is the lipid class that exhibits the most changes with age, suggesting that TAG metabolism is particularly sensitive to the aging process in mice. Collectively, our data show that the Lipidyzer platform provides comprehensive profiling of the most prevalent lipids in plasma in a simple and automated manner

'Where is the child I used to be?' Childhood remembered - Günter Grass’s The Tin Drum, Christa Wolf’s A Model Childhood and W.G. Sebald’s Austerlitz

Genes with reduced expression in Q 30 -YFP colonies versus Q 0 -YFP colonies. The table summarizes the expression differences of two data sets (Q0_3d, Q30_3d). Standard deviation and p-values were obtained as described above. Hits with a p-value greater than 0.05 are indicated in grey. (DOCX 16 kb

The Lid Domain of Caenorhabditis elegans Hsc70 Influences ATP Turnover, Cofactor Binding and Protein Folding Activity

Hsc70 is a conserved ATP-dependent molecular chaperone, which utilizes the energy of ATP hydrolysis to alter the folding state of its client proteins. In contrast to the Hsc70 systems of bacteria, yeast and humans, the Hsc70 system of C. elegans (CeHsc70) has not been studied to date

Cross-Platform Comparison of Untargeted and Targeted Lipidomics Approaches on Aging Mouse Plasma.

Lipidomics - the global assessment of lipids - can be performed using a variety of mass spectrometry (MS)-based approaches. However, choosing the optimal approach in terms of lipid coverage, robustness and throughput can be a challenging task. Here, we compare a novel targeted quantitative lipidomics platform known as the Lipidyzer to a conventional untargeted liquid chromatography (LC)-MS approach. We find that both platforms are efficient in profiling more than 300 lipids across 11 lipid classes in mouse plasma with precision and accuracy below 20% for most lipids. While the untargeted and targeted platforms detect similar numbers of lipids, the former identifies a broader range of lipid classes and can unambiguously identify all three fatty acids in triacylglycerols (TAG). Quantitative measurements from both approaches exhibit a median correlation coefficient (r) of 0.99 using a dilution series of deuterated internal standards and 0.71 using endogenous plasma lipids in the context of aging. Application of both platforms to plasma from aging mouse reveals similar changes in total lipid levels across all major lipid classes and in specific lipid species. Interestingly, TAG is the lipid class that exhibits the most changes with age, suggesting that TAG metabolism is particularly sensitive to the aging process in mice. Collectively, our data show that the Lipidyzer platform provides comprehensive profiling of the most prevalent lipids in plasma in a simple and automated manner

CeHsc70 truncation mutants show an altered ATP turnover.

<p>(<b>A</b>) Domain organization and amino acid identity (Id) and homology (Hom) of CeHsc70 towards bacterial, yeast and human homologs. The truncation mutants generated in this work are indicated by black arrows. (<b>B</b>) Structure of DnaK based on the PDB file 2KHO <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033980#pone.0033980-Bertelsen1" target="_blank">[93]</a>. The truncations are colored in red (CeHsc70-Δ384), red and blue (CeHsc70-Δ512) and red, blue and yellow (CeHsc70-Δ545). The lid region, which is missing in the CeHsc70-Δ545 mutant, is highlighted in orange. (<b>C</b>) The single-turnover experiments using 20 µM CeHsc70 variants were performed as outlined in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033980#s4" target="_blank">Materials and Methods</a> section in standard buffer at 25°C. Data for CeHsc70-Δ384 (▾), CeHsc70-Δ512 (▴), CeHsc70-Δ545 (•) and CeHsc70 (▪) were fit to single exponential functions. The inset shows the initial phase of the hydrolysis reaction within the first 200 s.</p

Enzymatic parameters of cofactor interactions with lid domain mutants.

<p>ATPase activities were determined in standard buffer as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033980#s4" target="_blank">Materials and Methods</a> section. DNJ-13 stimulation or BAG-1 inhibition were not observed in some experiments (denoted by “n.d.”). Consequently an apparent K_D cannot be deduced. The semi-quantitative value “tight” points to the fact that in the respective experiment, quantitative binding appeared substoichiometric. Consequently, no reasonable data fitting can be performed, using the normal absorption isotherm. K_D denotes the apparent affinity. The errors represent standard deviations of three independent experiments.</p

DNJ-13 interacts with CeHsc70 in presence of ATP and is released by BAG-1.

<p>(<b>A</b>) Domain organization of DNJ-13 homologs from <i>C. elegans</i>, <i>E. coli</i>, <i>S. cerevisiae</i> and <i>H. sapiens</i>. Percentages relate to identical (Id) and homolog (Hom) residues in respect to the nematode protein. (<b>B</b>) Steady-state ATPase activities were measured in the presence of increasing amounts of DNJ-13 for either CeHsc70 (•), CeHsc70-Δ512 (○) or CeHsc70-Δ545 (▾). Data were analyzed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033980#s4" target="_blank">Materials and Methods</a> section. (<b>C</b>) Single-turnover measurements of 10 µM CeHsc70-Δ384 (∇), CeHsc70-Δ512 (○), CeHsc70-Δ545 (▾) and CeHsc70 (•) in the presence of 15 µM DNJ-13. All data points were fit to single exponential functions. (<b>D</b>) dc/dt plots were generated from sedimentation velocity experiments of 300 nM *DNJ-13 in the absence (black) or in the presence of 3 µM CeHsc70 (pink). The influence of nucleotides was analyzed by addition of 4 mM of either ADP (gold), AMP-PNP (red), ATPγS (turqoise) or ATP (blue) to 300 nM *DNJ-13 and 3 µM CeHsc70. (<b>E</b>) dc/dt profiles of sedimentation velocity experiments of 300 nM *DNJ-13 in the presence of either 3 µM CeHsc70 (blue), CeHsc70-Δ384 (red), CeHsc70-Δ512 (black) or CeHsc70-Δ545 (green) in the presence of ATP.</p

The ternary interaction of CeHsc70 with BAG-1 and DNJ-13 is affected by the lid domain truncations.

<p>(<b>A</b>) dc/dt plots were generated from sedimentation velocity experiments of 300 nM *DNJ-13 in the absence (black) or in the presence of 3 µM CeHsc70 and 4 mM ATP (blue). The influence of BAG-1 on complex formation was analyzed by addition of 15 µM BAG-1 to *DNJ-13-CeHsc70-ATP (green). (<b>B</b>) The ATPase activity of 1 µM CeHsc70 (•), CeHsc70-Δ512 (○) or CeHsc70-Δ545 (▾) was measured with increasing amounts of DNJ-13 in the presence of 2 µM BAG-1 in standard buffer at 25°C. Data analysis was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033980#s4" target="_blank">Materials and Methods</a>.</p

Lid domain truncations reduce the refolding ability of CeHsc70.

<p>(<b>A</b>) Kinetics of firefly luciferase refolding in the presence of different chaperone combinations: CeHsc70/DNJ-13/BAG-1 (∇), CeHsc70/DNJ-13 (▪), CeHsc70/BAG-1 (♦), CeHsc70 (○), BAG-1 (□) and DNJ-13 (▾). Additionally the luminescence of a sample without chaperones and cofactors was analyzed (▴). Protein concentrations were 3.2 µM CeHsc70, 0.8 µM DNJ-13 and 0.4 µM BAG-1. Luciferase refolding assays were carried out as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033980#s4" target="_blank">Materials and Methods</a>. (<b>B</b>) Steady-state ATPase activities (black squares, left ordinate) and luciferase refolding efficiency (blue circles, right ordinate) were determined for 3.2 µM CeHsc70 and 0.8 µM DNJ-13 at different BAG-1 concentrations under standard conditions. (<b>C</b>) The luciferase refolding activity of either CeHsc70 (▪), CeHsc70-Δ545 (▾), CeHsc70-Δ512 (○) or CeHsc70-Δ384 (Δ) was determined in the presence of DNJ-13 and BAG-1. Additionally a control without chaperones and cofactors (◊) was recorded.</p