26 research outputs found

    Proteome-Wide Analysis of N‑Glycosylation Stoichiometry Using SWATH Technology

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    N-glycosylation is a crucial post-translational modification (PTM) and plays essential roles in biological processes. Several methods have been developed for the relative quantification of N-glycosylation at the proteome scale. However, the proportion of N-glycosylated forms in a total protein population, or the “N-glycosylation stoichiometry”, varies greatly among proteins or cellular states and is frequently missing due to the lack of robust technologies. In the present study, we developed a data-independent acquisition (DIA)-based strategy that enabled the in-depth measurement of N-glycosylation stoichiometry. A spectral library containing 3,509 N-glycosylated peptides and 17,525 fragment ions from human embryonic kidney cells 293 (HEK-293) cells was established from which the stoichiometries of 1,186 N-glycosites were calculated. These stoichiometric values differ greatly among different glycosites, and many glycosites tend to occur with low stoichiometry. We then investigated the N-glycosylation changes induced by tunicamycin in HEK-293 cells and by a temperature shift in Chinese hamster ovary (CHO) cells. Quantifying the proteome, N-glycoproteome, and N-glycosylation stoichiometry demonstrated that the regulation of N-glycosylation is primarily achieved by adjusting the N-glycosylation stoichiometry. In total, the stoichiometries of 2,274 glycosites were determined in the current study. Notably, our approach can be applied to other biological systems and other types of PTMs

    Quantitative Proteomic Analysis Identifies Targets and Pathways of a 2‑Aminobenzamide HDAC Inhibitor in Friedreich’s Ataxia Patient iPSC-Derived Neural Stem Cells

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    Members of the 2-aminobenzamide class of histone deacetylase (HDAC) inhibitors show promise as therapeutics for the neurodegenerative diseases Friedreich’s ataxia (FRDA) and Huntington’s disease (HD). While it is clear that HDAC3 is one of the important targets of the 2-aminobenzamide HDAC inhibitors, inhibition of other class I HDACs (HDACs 1 and 2) may also be involved in the beneficial effects of these compounds in FRDA and HD, and other HDAC interacting proteins may be impacted by the compound. To this end, we synthesized activity-based profiling probe (ABPP) versions of one of our HDAC inhibitors (compound 106), and in the present study we used a quantitative proteomic method coupled with multidimensional protein identification technology (MudPIT) to identify the proteins captured by the ABPP 106 probe. Nuclear proteins were extracted from FRDA patient iPSC-derived neural stem cells, and then were reacted with control and ABPP 106 probe. After reaction, the bound proteins were digested on the beads, and the peptides were modified using stable isotope-labeled formaldehyde to form dimethyl amine. The selectively bound proteins determined by mass spectrometry were subjected to functional and pathway analysis. Our findings suggest that the targets of compound 106 are involved not only in transcriptional regulation but also in posttranscriptional processing of mRNA

    Brain Proteome Changes Induced by Olfactory Learning in <i>Drosophila</i>

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    For more than 30 years, the study of learning and memory in Drosophila melanogaster (fruit fly) has used an olfactory learning paradigm and has resulted in the discovery of many genes involved in memory formation. By varying learning programs, the creation of different memory types can be achieved, from short-term memory formation to long-term. Previous studies in the fruit fly used gene mutation methods to identify genes involved in memory formation. Presumably, memory creation involves a combination of genes, pathways, and neural circuits. To examine memory formation at the protein level, a quantitative proteomic analysis was performed using olfactory learning and <sup>15</sup>N-labeled fruit flies. Differences were observed in protein expression and relevant pathways between different learning programs. Our data showed major protein expression changes occurred between short-term memory (STM) and long-lasting memory, and only minor changes were found between long-term memory (LTM) and anesthesia-resistant memory (ARM)

    Analysis of expression differences by MS analysis.

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    <p><b>A</b>) ESI mass spectrum of the <sup>14</sup>N LAB (red) and <sup>15</sup>N HAB (blue) isotope forms of the GLO1 peptide GFGHIGIAVPDVYSACK in cerebellar tissue. The isotopologue patterns of the <sup>14</sup>N and <sup>15</sup>N peptide signals (m/z) were used for relative quantification with ProRata. <b>B</b>) Extracted ion chromatograms for the GLO1 peptide, extracted from A, showing an upregulation of GLO1 in LAB mice. No smoothing was applied.</p

    Incorporation rate of <sup>15</sup>N in (A) plasma and (B) brain proteins.

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    <p>In 5, 14 and 28 day old animals (PND 5, 14, 28) bacteria diet feeding provides a significantly higher incorporation of <sup>15</sup>N in plasma proteins compared to blue-green algae diet; only at the age of 56 days, blue-green algae fed diet animals show the same <sup>15</sup>N incorporation. In the cerebellum, a faster incorporation in adolescence at PND 14 reached significance (**p<0.01; *p<0.05).</p

    Food consumption of algae and bacteria diets of HAB/NAB/LAB dams and pups before weaning.

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    <p>Consumption of blue-green algae diet of HAB dams and their pups and consumption of bacteria diet of HAB/NAB/LAB dams and their pups before weaning (post natal days (PND) 1–24); <sup>14</sup>N diet 14N; <sup>15</sup>N-enriched diet.</p

    Body weight after weaning of animals fed with bacteria, blue-green algae and standard diet.

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    <p>Compared to standard fed HAB animals (orange dotted line), blue-green algae diet fed animals were significantly heavier (green line) and bacteria fed animals lighter (red line) (**p<0.01 blue-green algae vs. control ++p<0.01 bacteria vs. control).</p

    Depression-like behavior in the tail suspension test (TST).

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    <p>High (HAB), normal (NAB) and low (LAB) anxious animals were fed with <sup>14</sup>N and/or <sup>15</sup>N enriched bacteria diets. <b>A</b>) No behavioral changes due to diet <i>per se</i> were found for animals fed with bacteria diet compared to the animals of the respective lines of the HAB/NAB/LAB standard breeding of the same generation (dotted lines and indicated in <b>B</b>) as percentage difference of the standard breeding). However, <sup>15</sup>N fed HAB animals showed a strongly reduced immobility, indicating depression-like behavior, compared to <sup>14</sup>N fed HABs (**p<0.01). B) This differed significantly from the standard HAB/NAB/LAB breeding (*p<0.05). Due to division by zero, no value is given for <sup>14</sup>N fed LAB animals in relation to the standard breeding: values are <sup>14</sup>N LAB 0.53±0.3 sec vs. standard LAB 4.06±1.5 sec.</p

    Depression-like behavior of HAB animals in the tail suspension test (TST).

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    <p>Animals fed with blue-green algae diet hat a strongly reduced immobility, indicating depression-like behavior, compared to standard fed animals. This effect was even more pronounced in animals fed with the <sup>15</sup>N isotope (*p<0.05; **p<0.01).</p
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