Robust, Sensitive, and Automated Phosphopeptide Enrichment Optimized for Low Sample Amounts Applied to Primary Hippocampal Neurons

Because of the low stoichiometry of protein phosphorylation, targeted enrichment prior to LC–MS/MS analysis is still essential. The trend in phosphoproteome analysis is shifting toward an increasing number of biological replicates per experiment, ideally starting from very low sample amounts, placing new demands on enrichment protocols to make them less labor-intensive, more sensitive, and less prone to variability. Here we assessed an automated enrichment protocol using Fe(III)-IMAC cartridges on an AssayMAP Bravo platform to meet these demands. The automated Fe(III)-IMAC-based enrichment workflow proved to be more effective when compared to a TiO2-based enrichment using the same platform and a manual Ti(IV)-IMAC-based enrichment workflow. As initial samples, a dilution series of both human HeLa cell and primary rat hippocampal neuron lysates was used, going down to 0.1 μg of peptide starting material. The optimized workflow proved to be efficient, sensitive, and reproducible, identifying, localizing, and quantifying thousands of phosphosites from just micrograms of starting material. To further test the automated workflow in genuine biological applications, we monitored EGF-induced signaling in hippocampal neurons, starting with only 200 000 primary cells, resulting in ∼50 μg of protein material. This revealed a comprehensive phosphoproteome, showing regulation of multiple members of the MAPK pathway and reduced phosphorylation status of two glutamate receptors involved in synaptic plasticity

Robust, Sensitive, and Automated Phosphopeptide Enrichment Optimized for Low Sample Amounts Applied to Primary Hippocampal Neurons

Because of the low stoichiometry of protein phosphorylation, targeted enrichment prior to LC–MS/MS analysis is still essential. The trend in phosphoproteome analysis is shifting toward an increasing number of biological replicates per experiment, ideally starting from very low sample amounts, placing new demands on enrichment protocols to make them less labor-intensive, more sensitive, and less prone to variability. Here we assessed an automated enrichment protocol using Fe(III)-IMAC cartridges on an AssayMAP Bravo platform to meet these demands. The automated Fe(III)-IMAC-based enrichment workflow proved to be more effective when compared to a TiO2-based enrichment using the same platform and a manual Ti(IV)-IMAC-based enrichment workflow. As initial samples, a dilution series of both human HeLa cell and primary rat hippocampal neuron lysates was used, going down to 0.1 μg of peptide starting material. The optimized workflow proved to be efficient, sensitive, and reproducible, identifying, localizing, and quantifying thousands of phosphosites from just micrograms of starting material. To further test the automated workflow in genuine biological applications, we monitored EGF-induced signaling in hippocampal neurons, starting with only 200 000 primary cells, resulting in ∼50 μg of protein material. This revealed a comprehensive phosphoproteome, showing regulation of multiple members of the MAPK pathway and reduced phosphorylation status of two glutamate receptors involved in synaptic plasticity

Monitoring light/dark association dynamics of multi-protein complexes in cyanobacteria using size exclusion chromatography-based proteomics

Diurnal rhythms are recurring 24h patterns such as light/dark cycles that affect many natural environmental and biological processes. The cyanobacterium Synechococcus elongatus PCC 7942 (S. elongatus) produces its energy through photosynthesis and therefore its internal molecular machinery is strongly influenced by these diurnal rhythms. Moreover, it has one of the simplest, self-sustained, circadian rhythms, extensively studied functionally and structurally. These characteristics together with the relatively small genome of S. elongatus, make it an ideal model system for the study of diurnal and circadian rhythms. Although expression of many gene transcripts has been shown to fluctuate in phase with the circadian rhythm, fluctuations at the protein level were less pronounced. This led us to hypothesize that the diurnal adaptation occurs at the level of higher organization of protein complexes. Therefore, we probed the abundance and constituency of S. elongatus protein complexes during the day and night. Following several well-known complexes such as the RNA polymerase, the ribosome and photosynthetic protein complexes, we observe for the first time that these complexes change not only in abundance but also in constituency. Therefore, we conclude that the dynamic assembly of protein complexes is indeed also a key-player in the processes governing the diurnal rhythm. SIGNIFICANCE: The succession of day and night periods imposes drastic changes in all living organisms. Cyanobacteria produce their energy through photosynthesis and are therefore strongly influenced by diurnal rhythms. The cyanobacteria, Synechococcus elongatus PCC 7942 (S. elongatus), also exhibit a self-sustained biological clock. The connection between the central circadian oscillator and its output to the rest of the cell is not completely known. It has been shown that the expression of many gene transcripts heavily fluctuates in phase with the circadian rhythm; however, our recent global proteomics investigation revealed that the diurnal fluctuations seemed to be less pronounced at the protein level. As many known regulatory functions depend on protein-protein interactions (PPIs) and/or protein assemblies and the fact that so few fluctuations in protein abundances were observed earlier, here we investigated the diurnal adaptation at the level of dynamic changes in protein assembly. The paper demonstrates that the combination of native protein complex fractionation and high-resolution proteomics provides insight in the regulation of megadalton protein assemblies in cyanobacteria, including the ribosomal and photosynthetic complexes. The differences observed between the light and dark conditions in these complexes indicate a cyclic regulation of essential cellular processes

The effect of stress on core and peripheral body temperature in humans

<p>Even though there are indications that stress influences body temperature in humans, no study has systematically investigated the effects of stress on core and peripheral body temperature. The present study therefore aimed to investigate the effects of acute psychosocial stress on body temperature using different readout measurements. In two independent studies, male and female participants were exposed to a standardized laboratory stress task (the Trier Social Stress Test, TSST) or a non-stressful control task. Core temperature (intestinal and temporal artery) and peripheral temperature (facial and body skin temperature) were measured. Compared to the control condition, stress exposure decreased intestinal temperature but did not affect temporal artery temperature. Stress exposure resulted in changes in skin temperature that followed a gradient-like pattern, with decreases at distal skin locations such as the fingertip and finger base and unchanged skin temperature at proximal regions such as the infraclavicular area. Stress-induced effects on facial temperature displayed a sex-specific pattern, with decreased nasal skin temperature in females and increased cheek temperature in males. In conclusion, the amplitude and direction of stress-induced temperature changes depend on the site of temperature measurement in humans. This precludes a direct translation of the preclinical stress-induced hyperthermia paradigm, in which core temperature uniformly rises in response to stress to the human situation. Nevertheless, the effects of stress result in consistent temperature changes. Therefore, the present study supports the inclusion of body temperature as a physiological readout parameter of stress in future studies.</p>

Temporal Quantitative Proteomics of mGluR-induced Protein Translation and Phosphorylation in Neurons

At neuronal synapses, activation of group I metabotropic glutamate receptors (mGluR1/5) triggers a form of long-term depression (mGluR-LTD) that relies on new protein synthesis and the internalization of AMPA-type glutamate receptors. Dysregulation of these processes has been implicated in the development of mental disorders such as autism spectrum disorders and therefore merit a better understanding on a molecular level. Here, to study mGluR-induced signaling pathways, we integrated quantitative phosphoproteomics with the analyses of newly synthesized proteins via bio-orthogonal amino acids (azidohomoalanine) in a pulsed labeling strategy in cultured hippocampal neurons stimulated with DHPG, a specific agonist for group I mGluRs. We identified several kinases with important roles in DHPG-induced mGluR activation, which we confirmed using small molecule kinase inhibitors. Furthermore, changes in the AMPA receptor endocytosis pathway in both protein synthesis and protein phosphorylation were identified, whereby Intersectin-1 was validated as a novel player in this pathway. This study revealed several new insights into the molecular pathways downstream of group I mGluR activation in hippocampal neurons, and provides a rich resource for further analyses

Measurement of alcohol hangover severity:development of the Alcohol Hangover Severity Scale (AHSS)

<p>This study aims to develop a new alcohol hangover symptom severity scale and compare its effectiveness with the Hangover Symptoms Scale (HSS), the Acute Hangover Scale (AHS), and a one-item hangover score.</p><p>Data from 1,410 Dutch students (Penning et al., Alcohol Alcohol 47:248-252, 2012) on the severity of 47 hangover symptoms were re-analyzed to develop the Alcohol Hangover Severity Scale (AHSS). The psychometric properties of the AHSS were compared with those of the HSS and the AHS. A survey among 1,000 students compared the AHSS and HSS with a one-item hangover severity score. The AHSS was further tested in a naturalistic hangover experiment.</p><p>The 12 items of the AHSS were fatigue, clumsiness, dizziness, apathy, sweating, shivering, nausea, heart pounding, confusion, stomach pain, concentration problems, and thirst. The Penning et al. (Alcohol Alcohol 47:248-252, 2012) data revealed that the predictive validity of the AHSS (92.4 %) for the overall hangover score was significantly higher than that of the HSS (81.5 %) and the AHS (71.0 %). The survey data (N = 966) showed that scores on the AHSS (39.7 %) and the HSS (47.6 %) only moderately predicted the one-item hangover score. A total of 119 subjects completed the naturalistic study. On average, they consumed 9.7 alcoholic consumptions, yielding a mean estimated blood alcohol concentration (BAC) of 0.16 %. During hangover, the AHSS score correlated significantly with the number of alcoholic consumptions (r = 0.38, p <0.0001) and estimated BAC (r = 0.40, p <0.0001).</p><p>The AHS, HSS, and AHSS all seem appropriate for application in hangover research. The use of a one-item hangover scale is not recommended.</p>