Cryo-EM structure of metazoan TRAPPIII, the multi-subunit complex that activates the GTPase Rab1.

The TRAPP complexes are nucleotide exchange factors that play essential roles in membrane traffic and autophagy. TRAPPII activates Rab11, and TRAPPIII activates Rab1, with the two complexes sharing a core of small subunits that affect nucleotide exchange but being distinguished by specific large subunits that are essential for activity in vivo. Crystal structures of core subunits have revealed the mechanism of Rab activation, but how the core and the large subunits assemble to form the complexes is unknown. We report a cryo-EM structure of the entire Drosophila TRAPPIII complex. The TRAPPIII-specific subunits TRAPPC8 and TRAPPC11 hold the catalytic core like a pair of tongs, with TRAPPC12 and TRAPPC13 positioned at the joint between them. TRAPPC2 and TRAPPC2L link the core to the two large arms, with the interfaces containing residues affected by disease-causing mutations. The TRAPPC8 arm is positioned such that it would contact Rab1 that is bound to the core, indicating how the arm could determine the specificity of the complex. A lower resolution structure of TRAPPII shows a similar architecture and suggests that the TRAPP complexes evolved from a single ur-TRAPP.

SLC25A46 promotes mitochondrial fission and mediates resistance to lipotoxic stress in INS-1E insulin-secreting cells.

Glucose sensing in pancreatic ß-cells depends on oxidative phosphorylation and mitochondria-derived signals that promote insulin secretion. Using mass spectrometry-based phosphoproteomics to search for downstream effectors of glucose-dependent signal transduction in INS-1E insulinoma cells, we identified the outer mitochondrial membrane protein SLC25A46. Under resting glucose concentrations, SLC25A46 was phosphorylated on a pair of threonine residues (T44/T45) and was dephosphorylated in response to glucose-induced Ca2+ signals. Overexpression of SLC25A46 in INS-1E cells caused complete mitochondrial fragmentation, resulting in a mild mitochondrial defect associated with lowered glucose-induced insulin secretion. In contrast, inactivation of the Slc25a46 gene resulted in dramatic mitochondrial hyperfusion, without affecting respiratory activity or insulin secretion. Consequently, SLC25A46 is not essential for metabolism-secretion coupling under normal nutrient conditions. Importantly, insulin-secreting cells lacking SLC25A46 had an exacerbated sensitivity to lipotoxic conditions, undergoing massive apoptosis when exposed to palmitate. Therefore, in addition to its role in mitochondrial dynamics, SLC25A46 plays a role in preventing mitochondria-induced apoptosis in INS-E cells exposed to nutrient stress. By protecting mitochondria, SLC25A46 might help to maintain ß-cell mass essential for blood glucose control.

Biofungicides Based on Plant Extracts: On the Road to Organic Farming.

Phytopathogenic fungi are responsible for diseases in commercially important crops and cause major supply problems in the global food chain. Plants were able to protect themselves from disease before humans played an active role in protecting plants. They are known to synthesize a variety of secondary metabolites (SMs), such as terpenes, alkaloids, and phenolic compounds, which can be extracted using conventional and unconventional techniques to formulate biofungicides; plant extracts have antifungal activity and various mechanisms of action against these organisms. In addition, they are considered non-phytotoxic and potentially effective in disease control. They are a sustainable and economically viable alternative for use in agriculture, which is why biofungicides are increasingly recognized as an attractive option to solve the problems caused by synthetic fungicides. Currently, organic farming continues to grow, highlighting the importance of developing environmentally friendly alternatives for crop production. This review provides a compilation of the literature on biosynthesis, mechanisms of action of secondary metabolites against phytopathogens, extraction techniques and formulation of biofungicides, biological activity of plant extracts on phytopathogenic fungi, regulation, advantages, disadvantages and an overview of the current use of biofungicides in agriculture.

The Application of Ultrasound in Honey: Antioxidant Activity, Inhibitory Effect on α-amylase and α-glucosidase, and In Vitro Digestibility Assessment.

In the present study, the effects of ultrasound (10, 20, and 30 min) on the bioactive compounds, antioxidant capacity, enzymatic inhibition, and in vitro digestion of six honey extracts from the Oaxaca state, Mexico, were analyzed. Significant differences were found in each honey extract with respect to the ultrasonic treatment applied (p < 0.05). In the honey extract P-A1 treated with 20 min of ultrasound, the phenols reached a maximum concentration of 29.91 ± 1.56 mg EQ/100 g, and the flavonoids of 1.92 ± 0.01 mg EQ/100 g; in addition, an inhibition of α-amylase of 37.14 ± 0.09% was noted. There were also differences in the phases of intestinal and gastric digestion, presenting a decrease in phenols (3.92 ± 0.042 mg EQ/100 g), flavonoids (0.61 ± 0.17 mg EAG/100 mg), antioxidant capacity (8.89 ± 0.56 mg EAG/100 mg), and amylase inhibition (9.59 ± 1.38%). The results obtained from this study indicate that, in some honeys, the processing method could increase the concentration of bioactive compounds, the antioxidant capacity, and the enzymatic inhibition; however, when subjected to in vitro digestion, the properties of honey are modified. The results obtained could aid in the development of these compounds for use in traditional medicine as a natural source of bioactive compounds.

Proteomics of Human Milk: Definition of a Discovery Workflow for Clinical Research Studies.

Milk is a complex biological fluid composed mainly of water, carbohydrates, lipids, proteins, and diverse bioactive factors. Human milk represents a unique tailored source of nutrients that adapts during lactation to the specific needs of the developing infant. Proteins in milk have been studied for decades, and proteomics, peptidomics, and glycoproteomics are the main approaches previously deployed to decipher the proteome of human milk. In the present work, we aimed at implementing a highly automated pipeline for the proteomic analysis of human milk with liquid chromatography mass spectrometry (MS). Commercial human milk samples were used to evaluate and optimize workflows. Centrifugation for defatting milk samples was assessed before and after reduction, alkylation, and enzymatic digestion of proteins, without and with presence of surfactants. Skimmed milk samples were analyzed using isobaric labeling-based quantitative MS on an Orbitrap Tribrid mass spectrometer. Sample fractionation using isoelectric focusing was also evaluated to more deeply profile the human milk proteome. Finally, the most appropriate workflow was transferred to a liquid handling workstation for automated sample preparation. In conclusion, we have defined and describe herein an efficient highly automated proteomic workflow for human milk sample analysis. It is compatible with clinical research, possibly allowing the analysis of sufficiently large cohorts of samples.

The Addition of Microencapsulated or Nanoemulsified Bioactive Compounds Influences the Antioxidant and Antimicrobial Activities of a Fresh Cheese.

The objective of this study was to compare the effects of the incorporation of microcapsules or nanoemulsions with Opuntiaoligacantha on the quality of fresh cheese. Three treatments were established: Control, cheese with microcapsules (Micro), and cheese with nanoemulsion (Nano). The parameters evaluated were physicochemical (moisture, ash, fat, proteins, and pH), microbiological (mesophilic aerobic bacteria, mold-yeast, and total coliforms), functional (total phenols, flavonoids, and antioxidant capacity), and texture (hardness, elasticity, cohesion, and chewiness) during storage for 45 days at 4 °C. The results showed that adding microcapsules and nanoemulsion did not affect the physicochemical parameters of the cheese. Total coliforms decreased in all samples from the first days of storage (Control: 4.23 ± 0.12, Micro: 3.27 ± 0.02, and Nano: 2.68 ± 0.08 Log10 CFU), as well as aerobic mesophiles and mold-yeast counts. Regarding the functional properties, an increase in total phenols was observed in all treatments. The texture profile analysis showed that the addition of microcapsules and nanoemulsion influenced hardness (Control: 8.60 ± 1.12, Micro: 1.61 ± 0.31, and Nano: 3.27 ± 0.37 N). The antimicrobial effect was greater when nanoemulsions were added, while adding microcapsules influenced the antioxidant activity more positively.

Mitochondrial lysine deacetylation promotes energy metabolism and calcium signaling in insulin-secreting cells.

In pancreatic ß-cells, mitochondria generate signals that promote insulin granule exocytosis. Here we study how lysine acetylation of mitochondrial proteins mechanistically affects metabolism-secretion coupling in insulin-secreting cells. Using mass spectrometry-based proteomics, we identified lysine acetylation sites in rat insulinoma cell line clone 1E cells. In cells lacking the mitochondrial lysine deacetylase sirtuin-3 (SIRT3), several matrix proteins are hyperacetylated. Disruption of the SIRT3 gene has a deleterious effect on mitochondrial energy metabolism and Ca2+ signaling. Under resting conditions, SIRT3 deficient cells are overactivated, which elevates the respiratory rate and enhances calcium signaling and basal insulin secretion. In response to glucose, the SIRT3 knockout cells are unable to mount a sustained cytosolic ATP response. Calcium signaling is strongly reduced and the respiratory response as well as insulin secretion are blunted. We propose mitochondrial protein lysine acetylation as a control mechanism in ß-cell energy metabolism and Ca2+ signaling.-De Marchi, U., Galindo, A. N., Thevenet, J., Hermant, A., Bermont, F., Lassueur, S., Domingo, J. S., Kussmann, M., Dayon, L., Wiederkehr, A. Mitochondrial lysine deacetylation promotes energy metabolism and calcium signaling in insulin-secreting cells.

Proteomes of Paired Human Cerebrospinal Fluid and Plasma: Relation to Blood-Brain Barrier Permeability in Older Adults.

The systems-level relationship between the proteomes of cerebrospinal fluid (CSF) and plasma has not been comprehensively described so far. Recently developed shotgun proteomic workflows allow for deeper characterization of the proteomes from body fluids in much larger sample size. We deployed state-of-the-art mass spectrometry-based proteomics in paired CSF and plasma samples volunteered by 120 elders with and without cognitive impairment to comprehensively characterize and examine compartmental proteome differences and relationships between both body fluids. We further assessed the influence of blood-brain barrier (BBB) integrity and tested the hypothesis that BBB breakdown can be identified from CSF and plasma proteome alterations in nondemented elders. We quantified 790 proteins in CSF and 422 proteins in plasma, and 255 of the proteins were identified in both compartments. Pearson's statistics determined 28 proteins with associated levels between CSF and plasma. BBB integrity as defined with the CSF/serum albumin index influenced 76 CSF/plasma protein ratios. In least absolute shrinkage and selection operator models, CSF and plasma proteins improved identification of BBB impairment. In conclusion, we provide here a first comprehensive draft map of interacting human CSF and plasma proteomes, in view of their complex and dynamic compositions, and influence of the BBB.

Glucose-dependent phosphorylation signaling pathways and crosstalk to mitochondrial respiration in insulin secreting cells.

BACKGROUND: Glucose is the main secretagogue of pancreatic beta-cells. Uptake and metabolism of the nutrient stimulates the beta-cell to release the blood glucose lowering hormone insulin. This metabolic activation is associated with a pronounced increase in mitochondrial respiration. Glucose stimulation also initiates a number of signal transduction pathways for the coordinated regulation of multiple biological processes required for insulin secretion. METHODS: Shotgun proteomics including TiO2 enrichment of phosphorylated peptides followed by liquid chromatography tandem mass spectrometry on lysates from glucose-stimulated INS-1E cells was used to identify glucose regulated phosphorylated proteins and signal transduction pathways. Kinase substrate enrichment analysis (KSEA) was applied to identify key regulated kinases and phosphatases. Glucose-induced oxygen consumption was measured using a XF96 Seahorse instrument to reveal cross talk between glucose-regulated kinases and mitochondrial activation. RESULTS: Our kinetic analysis of substrate phosphorylation reveal the molecular mechanism leading to rapid activation of insulin biogenesis, vesicle trafficking, insulin granule exocytosis and cytoskeleton remodeling. Kinase-substrate enrichment identified upstream kinases and phosphatases and time-dependent activity changes during glucose stimulation. Activity trajectories of well-known glucose-regulated kinases and phosphatases are described. In addition, we predict activity changes in a number of kinases including NUAK1, not or only poorly studied in the context of the pancreatic beta-cell. Furthermore, we pharmacologically tested whether signaling pathways predicted by kinase-substrate enrichment analysis affected glucose-dependent acceleration of mitochondrial respiration. We find that phosphoinositide 3-kinase, Ca2+/calmodulin dependent protein kinase and protein kinase C contribute to short-term regulation of energy metabolism. CONCLUSIONS: Our results provide a global view into the regulation of kinases and phosphatases in insulin secreting cells and suggest cross talk between glucose-induced signal transduction and mitochondrial activation.

Deep Dive on the Proteome of Human Cerebrospinal Fluid: A Valuable Data Resource for Biomarker Discovery and Missing Protein Identification.

Cerebrospinal fluid (CSF) is a body fluid of choice for biomarker studies of brain disorders but remains relatively under-studied compared with other biological fluids such as plasma, partly due to the more invasive means of its sample collection. The present study establishes an in-depth CSF proteome through the analysis of a unique CSF sample from a pool of donors. After immunoaffinity depletion, the CSF sample was fractionated using off-gel electrophoresis and analyzed with liquid chromatography tandem mass spectrometry (MS) using the latest generation of hybrid Orbitrap mass spectrometers. The shotgun proteomic analysis allowed the identification of 20 689 peptides mapping on 3379 proteins. To the best of our knowledge, the obtained data set constitutes the largest CSF proteome published so far. Among the CSF proteins identified, 34% correspond to genes whose transcripts are highly expressed in brain according to the Human Protein Atlas. The principal Alzheimer's disease biomarkers (e.g., tau protein, amyloid-ß, apolipoprotein E, and neurogranin) were detected. Importantly, our data set significantly contributes to the Chromosome-centric Human Proteome Project (C-HPP), and 12 proteins considered as missing are proposed for validation in accordance with the HPP guidelines. Of these 12 proteins, 8 proteins are based on 2 to 6 uniquely mapping peptides from this CSF analysis, and 4 match a new peptide with a "stranded" single peptide in PeptideAtlas from previous CSF studies. The MS proteomic data are available to the ProteomeXchange Consortium ( http://www.proteomexchange.org/ ) with the data set identifier PXD009646.

Identification of Missing Proteins in Normal Human Cerebrospinal Fluid.

The cerebrospinal fluid (CSF) proteome data set presented herein was obtained after immunodepletion of abundant proteins and off-gel electrophoresis fractionation of a commercial pool of normal human CSF; liquid chromatography tandem mass spectrometry analysis was performed with a linear ion trap-Orbitrap Elite. We report the identification of 12 344 peptides mapping on 2281 proteins. In the context of the Chromosome-centric Human Proteome Project (C-HPP), the existence of seven missing proteins is proposed to be validated. This data set is available to the ProteomeXchange Consortium ( http://www.proteomexchange.org/ ) with the data set identifier PXD008029.

Systematic Evaluation of the Use of Human Plasma and Serum for Mass-Spectrometry-Based Shotgun Proteomics.

Over the last two decades, EDTA-plasma has been used as the preferred sample matrix for human blood proteomic profiling. Serum has also been employed widely. Only a few studies have assessed the difference and relevance of the proteome profiles obtained from plasma samples, such as EDTA-plasma or lithium-heparin-plasma, and serum. A more complete evaluation of the use of EDTA-plasma, heparin-plasma, and serum would greatly expand the comprehensiveness of shotgun proteomics of blood samples. In this study, we evaluated the use of heparin-plasma with respect to EDTA-plasma and serum to profile blood proteomes using a scalable automated proteomic pipeline (ASAP2). The use of plasma and serum for mass-spectrometry-based shotgun proteomics was first tested with commercial pooled samples. The proteome coverage consistency and the quantitative performance were compared. Furthermore, protein measurements in EDTA-plasma and heparin-plasma samples were comparatively studied using matched sample pairs from 20 individuals from the Australian Imaging, Biomarkers and Lifestyle (AIBL) Study. We identified 442 proteins in common between EDTA-plasma and heparin-plasma samples. Overall agreement of the relative protein quantification between the sample pairs demonstrated that shotgun proteomics using workflows such as the ASAP2 is suitable in analyzing heparin-plasma and that such sample type may be considered in large-scale clinical research studies. Moreover, the partial proteome coverage overlaps (e.g., ∼70%) showed that measures from heparin-plasma could be complementary to those obtained from EDTA-plasma.

An Adaptive Pipeline To Maximize Isobaric Tagging Data in Large-Scale MS-Based Proteomics.

Isobaric tagging is the method of choice in mass-spectrometry-based proteomics for comparing several conditions at a time. Despite its multiplexing capabilities, some drawbacks appear when multiple experiments are merged for comparison in large sample-size studies due to the presence of missing values, which result from the stochastic nature of the data-dependent acquisition mode. Another indirect cause of data incompleteness might derive from the proteomic-typical data-processing workflow that first identifies proteins in individual experiments and then only quantifies those identified proteins, leaving a large number of unmatched spectra with quantitative information unexploited. Inspired by untargeted metabolomic and label-free proteomic workflows, we developed a quantification-driven bioinformatic pipeline (Quantify then Identify (QtI)) that optimizes the processing of isobaric tandem mass tag (TMT) data from large-scale studies. This pipeline includes innovative features, such as peak filtering with a self-adaptive preprocessing pipeline optimization method, Peptide Match Rescue, and Optimized Post-Translational Modification. QtI outperforms a classical benchmark workflow in terms of quantification and identification rates, significantly reducing missing data while preserving unmatched features for quantitative comparison. The number of unexploited tandem mass spectra was reduced by 77 and 62% for two human cerebrospinal fluid and plasma data sets, respectively.

Coordinated activation of mitochondrial respiration and exocytosis mediated by PKC signaling in pancreatic ß cells.

Mitochondria play a central role in pancreatic ß-cell nutrient sensing by coupling their metabolism to plasma membrane excitability and insulin granule exocytosis. Whether non-nutrient secretagogues stimulate mitochondria as part of the molecular mechanism to promote insulin secretion is not known. Here, we show that PKC signaling, which is employed by many non-nutrient secretagogues, augments mitochondrial respiration in INS-1E (rat insulinoma cell line clone 1E) and human pancreatic ß cells. The phorbol ester, phorbol 12-myristate 13-acetate, accelerates mitochondrial respiration at both resting and stimulatory glucose concentrations. A range of inhibitors of novel PKC isoforms prevent phorbol ester-induced respiration. Respiratory response was blocked by oligomycin that demonstrated PKC-dependent acceleration of mitochondrial ATP synthesis. Enhanced respiration was observed even when glycolysis was bypassed or fatty acid transport was blocked, which suggested that PKC regulates mitochondrial processes rather than upstream catabolic fluxes. A phosphoproteome study of phorbol ester-stimulated INS-1E cells maintained under resting (2.5 mM) glucose revealed a large number of phosphorylation sites that were altered during short-term activation of PKC signaling. The data set was enriched for proteins that are involved in gene expression, cytoskeleton remodeling, secretory vesicle transport, and exocytosis. Interactome analysis identified PKC, C-Raf, and ERK1/2 as the central phosphointeraction cluster. Prevention of ERK1/2 signaling by using a MEK1 inhibitor caused a marked decreased in phorbol 12-myristate 13-acetate-induced mitochondrial respiration. ERK1/2 signaling module therefore links PKC activation to downstream mitochondrial activation. We conclude that non-nutrient secretagogues act, in part, via PKC and downstream ERK1/2 signaling to stimulate mitochondrial energy production to compensate for energy expenditure that is linked to ß-cell activation.-Santo-Domingo, J., Chareyron, I., Dayon, L., Galindo, A. N., Cominetti, O., Giménez, M. P. G., De Marchi, U., Canto, C., Kussmann, M., Wiederkehr, A. Coordinated activation of mitochondrial respiration and exocytosis mediated by PKC signaling in pancreatic ß cells.

Mutational analysis of the Aspergillus ambient pH receptor PalH underscores its potential as a target for antifungal compounds.

The pal/RIM ambient pH signalling pathway is crucial for the ability of pathogenic fungi to infect hosts. The Aspergillus nidulans 7-TMD receptor PalH senses alkaline pH, subsequently facilitating ubiquitination of the arrestin PalF. Ubiquitinated PalF triggers downstream signalling events. The mechanism(s) by which PalH transduces the alkaline pH signal to PalF is poorly understood. We show that PalH is phosphorylated in a signal dependent manner, resembling mammalian GPCRs, although PalH phosphorylation, in contrast to mammalian GPCRs, is arrestin dependent. A genetic screen revealed that an ambient-exposed region comprising the extracellular loop connecting TM4-TM5 and ambient-proximal residues within TM5 is required for signalling. In contrast, substitution by alanines of four aromatic residues within TM6 and TM7 results in a weak 'constitutive' activation of the pathway. Our data support the hypothesis that PalH mechanistically resembles mammalian GPCRs that signal via arrestins, such that the relative positions of individual helices within the heptahelical bundle determines the Pro316-dependent transition between inactive and active PalH conformations, governed by an ambient-exposed region including critical Tyr259 that potentially represents an agonist binding site. These findings open the possibility of screening for agonist compounds stabilizing the inactive conformation of PalH, which might act as antifungal drugs against ascomycetes.

High-throughput and simultaneous quantitative analysis of homocysteine-methionine cycle metabolites and co-factors in blood plasma and cerebrospinal fluid by isotope dilution LC-MS/MS.

The methionine cycle is a key pathway contributing to the regulation of human health, with well-established involvement in cardiovascular diseases and cognitive function. Changes in one-carbon cycle metabolites have also been associated with mild cognitive decline, vascular dementia, and Alzheimer's disease. Today, there is no single analytical method to monitor both metabolites and co-factors of the methionine cycle. To address this limitation, we here report for the first time a new method for the simultaneous quantitation of 17 metabolites in the methionine cycle, which are homocysteic acid, taurine, serine, cysteine, glycine, homocysteine, riboflavin, methionine, pyridoxine, cystathionine, pyridoxamine, S-adenosylhomocysteine, S-adenosylmethionine, betaine, choline, dimethylglycine, and 5-methyltetrahydrofolic acid. This multianalyte method, developed using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), provides a highly accurate and precise quantitation of these 17 metabolites for both plasma and cerebrospinal fluid metabolite monitoring. The method requires a simple sample preparation, which, combined with a short chromatographic run time, ensures a high sample throughput. This analytical strategy will thus provide a novel metabolomics approach to be employed in large-scale observational and intervention studies. We expect such a robust method to be particularly relevant for broad and deep molecular phenotyping of individuals in relation to their nutritional requirements, health monitoring, and disease risk management.

Proteomic Biomarker Discovery in 1000 Human Plasma Samples with Mass Spectrometry.

The overall impact of proteomics on clinical research and its translation has lagged behind expectations. One recognized caveat is the limited size (subject numbers) of (pre)clinical studies performed at the discovery stage, the findings of which fail to be replicated in larger verification/validation trials. Compromised study designs and insufficient statistical power are consequences of the to-date still limited capacity of mass spectrometry (MS)-based workflows to handle large numbers of samples in a realistic time frame, while delivering comprehensive proteome coverages. We developed a highly automated proteomic biomarker discovery workflow. Herein, we have applied this approach to analyze 1000 plasma samples from the multicentered human dietary intervention study "DiOGenes". Study design, sample randomization, tracking, and logistics were the foundations of our large-scale study. We checked the quality of the MS data and provided descriptive statistics. The data set was interrogated for proteins with most stable expression levels in that set of plasma samples. We evaluated standard clinical variables that typically impact forthcoming results and assessed body mass index-associated and gender-specific proteins at two time points. We demonstrate that analyzing a large number of human plasma samples for biomarker discovery with MS using isobaric tagging is feasible, providing robust and consistent biological results.

Aspergillus nidulans Ambient pH Signaling Does Not Require Endocytosis.

Aspergillus nidulans (Pal) ambient pH signaling takes place in cortical structures containing components of the ESCRT pathway, which are hijacked by the alkaline pH-activated, ubiquitin-modified version of the arrestin-like protein PalF and taken to the plasma membrane. There, ESCRTs scaffold the assembly of dedicated Pal proteins acting downstream. The molecular details of this pathway, which results in the two-step proteolytic processing of the transcription factor PacC, have received considerable attention due to the key role that it plays in fungal pathogenicity. While current evidence strongly indicates that the pH signaling role of ESCRT complexes is limited to plasma membrane-associated structures where PacC proteolysis would take place, the localization of the PalB protease, which almost certainly catalyzes the first and only pH-regulated proteolytic step, had not been investigated. In view of ESCRT participation, this formally leaves open the possibility that PalB activation requires endocytic internalization. As endocytosis is essential for hyphal growth, nonlethal endocytic mutations are predicted to cause an incomplete block. We used a SynA internalization assay to measure the extent to which any given mutation prevents endocytosis. We show that none of the tested mutations impairing endocytosis to different degrees, including slaB1, conditionally causing a complete block, have any effect on the activation of the pathway. We further show that PalB, like PalA and PalC, localizes to cortical structures in an alkaline pH-dependent manner. Therefore, signaling through the Pal pathway does not involve endocytosis.

Proteomics of Cerebrospinal Fluid: Throughput and Robustness Using a Scalable Automated Analysis Pipeline for Biomarker Discovery.

Cerebrospinal fluid (CSF) is a body fluid of high clinical relevance and an important source of potential biomarkers for brain-associated damages, such as traumatic brain injury and stroke, and for brain diseases, such as Alzheimer's and Parkinson's. Herein, we have implemented, evaluated, and validated a scalable automated proteomic pipeline (ASAP(2)) for the sample preparation and proteomic analysis of CSF, enabling increased throughput and robustness for biomarker discovery. Human CSF samples were depleted from abundant proteins and subjected to automated reduction, alkylation, protein digestion, tandem mass tag (TMT) 6-plex labeling, pooling, and sample cleanup in a 96-well-plate format before reversed-phase liquid chromatography tandem mass spectrometry (RP-LC MS/MS). We showed the impact on the CSF proteome coverage of applying the depletion of abundant proteins, which is usually performed on blood plasma or serum samples. Using ASAP(2) to analyze 96 identical CSF samples, we determined the analytical figures of merit of our shotgun proteomic approach regarding proteome coverage consistency (i.e., 387 proteins), quantitative accuracy, and individual protein variability. We demonstrated that, as for human plasma samples, ASAP(2) is efficient in analyzing large numbers of human CSF samples and is a valuable tool for biomarker discovery. The data has been deposited to the ProteomeXchange with identifier PXD003024.

Comprehensive and Scalable Highly Automated MS-Based Proteomic Workflow for Clinical Biomarker Discovery in Human Plasma.

Over the past decade, mass spectrometric performance has greatly improved in terms of sensitivity, dynamic range, and speed. By contrast, only limited progress has been accomplished with regard to automation, throughput, and robustness of the proteomic sample preparation process upstream of mass spectrometry. The present work delivers an optimized analysis of human plasma samples in both small preclinical and large clinical studies, enabled by the development of a highly automated quantitative proteomic workflow. Several iterative evaluation and validation steps were performed before process "design freeze" and development completion. A robotic liquid handling workflow and platform (including reduction, alkylation, digestion, TMT labeling, pooling, and purification) were shown to provide better quantitative trueness and precision than manual operation at the bench. Depletion of the most abundant human plasma proteins and subsequent buffer exchange were also developed and integrated. Finally, 96 identical pooled human plasma samples were prepared in a 96-well plate format, and each sample was individually subjected to our developed workflow. This test revealed increased throughput and robustness compared with to-date published manual or less automated workflows. Our workflow is ready-to-use for future (pre-) clinical studies. We expect our work to facilitate, accelerate, and improve clinical proteomic discovery in human blood plasma.