Improved Methodology for Sensitive and Rapid Quantitative Proteomic Analysis of Adult‐Derived Mouse Microglia: Application to a Novel In Vitro Mouse Microglial Cell Model

Microglia, as the resident brain immune cells, can exhibit a broad range of activation phenotypes, which have been implicated in a multitude of central nervous system disorders. Current widely studied microglial cell lines are mainly derived from neonatal rodent brain that can limit their relevance to homeostatic function and disease‐related neuroimmune responses in the adult brain. Recently, an adult mouse brain‐derived microglial cell line has been established; however, a comprehensive proteome dataset remains lacking. Here, an optimization method for sensitive and rapid quantitative proteomic analysis of microglia is described that involves suspension trapping (S‐Trap) for efficient and reproducible protein extraction from a limited number of microglial cells expected from an adult mouse brain (≈300 000). Using a 2‐h gradient on a 75‐cm UPLC column with a modified data dependent acquisition method on a hybrid quadrupole‐Orbitrap mass spectrometer, 4855 total proteins have been identified where 4698 of which are quantifiable by label‐free quantitation with a median and average coefficient of variation (CV) of 6.7% and 10.6%, respectively. This dataset highlights the high depth of proteome coverage and related quantitation precision of the adult‐derived microglial proteome including proteins associated with several key pathways related to immune response. Data are available via ProteomeXchange with identifier PXD012006.     

Dl‐3‐n‐butylphthalide attenuates acute inflammatory activation in rats with spinal cord injury by inhibiting microglial TLR4/NF‐κB signalling

In this study, we examined the neuroprotective effects and anti‐inflammatory properties of Dl‐3‐n‐butylphthalide (NBP) in Sprague‐Dawley (SD) rats following traumatic spinal cord injury (SCI) as well as microglia activation and inflammatory response both in vivo and in vitro. Our results showed that NBP improved the locomotor recovery of SD rats after SCI an significantly diminished the lesion cavity area of the spinal cord, apoptotic activity in neurons, and the number of TUNEL‐positive cells at 7 days post‐injury. NBP inhibited activation of microglia, diminished the release of inflammatory mediators, and reduced the upregulation of microglial TLR4/NF‐κB expression at 1 day post‐injury. In a co‐culture system with BV‐2 cells and PC12 cells, NBP significantly reduced the cytotoxicity of BV‐2 cells following lipopolysaccharide (LPS) stimulation. In addition, NBP reduced the activation of BV‐2 cells, diminished the release of inflammatory mediators, and inhibited microglial TLR4/NF‐κB expression in BV‐2 cells. Our findings demonstrate that NBP may have neuroprotective and anti‐inflammatory properties in the treatment of SCI by inhibiting the activation of microglia via TLR4/NF‐κB signalling.     

Regulation of microglial migration,phagocytosis, and neurite outgrowth by HO‐1/CO signaling

Clearance of infected and apoptotic neuronal corpses during inflammatory conditions is a fundamental process to create a favorable environment for neuronal recovery. Microglia are the resident immune cells and the predominant phagocytic cells of the CNS, showing a multitude of cellular responses upon activation. Here, we investigated in functional assays how the CO generating enzyme heme oxygenase 1 (HO‐1) influences BV‐2 microglial migration, clearance of debris, and neurite outgrowth of human NT2 neurons. Stimulation of HO‐1 activity attenuated microglial migration in a scratch wound assay, and phagocytosis in a cell culture model of acute inflammation comprising lipopolysaccharide (LPS)‐activated microglia and apoptosis‐induced neurons. Application of a CO donor prevented the production of NO during LPS stimulation, and reduced microglial migration and engulfment of neuronal debris. LPS‐activated microglia inhibited neurite elongation of human neurons without requiring direct cell–cell surface contact. The inhibition of neurite outgrowth was totally reversed by application of exogenous CO or increased internal CO production through supply of the substrate hemin to HO. Our results point towards a vital cytoprotective role of HO‐1/CO signaling after microglial activation. In addition, they support a therapeutic potential of CO releasing chemical agents in the treatment of excessive inflammatory conditions in the CNS. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 854–876, 2015     

Targeted label‐free quantification of interleukin‐8 in PMA‐activated U937 cell secretome by nanoLC‐ESI‐MS/MS‐sSRM

Monocytes are a part of the innate immune system. Their differentiation into macrophages changes their cellular proteome and secretome. Particularly secretome components such as cytokines are crucial for immune response and inflammation in many diseases. Differentiation of human lymphoma cell line U937 can be triggered by phorbol 12‐myristate 13‐acetate (PMA). Screening of the cytokine release in U937 upon PMA stimulation by cytometric bead array almost exclusively showed interleukin‐8 (IL‐8). Next, a label‐free nanoLC‐ESI‐MS/MS‐sSRM method for quantification of IL‐8 in the cell secretome was established and applied to monitor the time kinetics of PMA treatment in different concentrations. Targeted secretome analysis was achieved by scheduled SRM‐MS using one proteotypic peptide as precursor ion and four mass transitions. Label‐free quantification was performed by external calibration using IL‐8 standard. Validation results indicated that the method was suited for the quantification of IL‐8 in the secretome. The maximal IL‐8 release of 62.4 ng/mL was observed after incubating cells treated by 50 ng/mL PMA for 48 h. The method can now be used for quantification of IL‐8 release from different cells under various conditions. Furthermore, it can be easily expanded to other secreted proteins detected by untargeted screening methods.     

Proteomic analysis of mouse astrocytes and their secretome by a combination of FASP and StageTip‐based,high pH,reversed‐phase fractionation

Astrocytes are the most abundant cells in the CNS, but their function remains largely unknown. Characterization of the whole‐cell proteome and secretome in astrocytes would facilitate the study of their functions in various neurodegenerative diseases and astrocyte–neuron communication. To build a reference proteome, we established a C8‐D1A astrocyte proteome to a depth of 7265 unique protein groups using a novel strategy that combined two‐step digestion, filter‐aided sample preparation, StageTip‐based high pH fractionation, and high‐resolution MS. Nearly, 6000 unique protein groups were identified from conditioned media of astrocyte cultures, constituting the largest astrocyte secretome that has been reported. High‐confidence whole‐cell proteomes and secretomes are valuable resources in studying astrocyte function by label‐free quantitation and bioinformatics analysis. All MS data have been deposited in the ProteomeXchange with identifier PXD000501 ( http://proteomecentral.proteomexchange.org/dataset/PXD000501 ).     

Interleukin‐12 P40 induces the expression of TNF‐α in microglia and macrophages

Recently, it has been found that overproduction of IL‐12 can be dangerous to the host as it is involved in the pathogenesis of a number of autoimmune inflammatory diseases such as multiple sclerosis. It is composed of two different subunits – p40 and p35. Expression of p40 mRNA but not that of p35 mRNA in excessive amount in the CNS of patients with Multiple Sclerosis (MS) suggests that IL‐12 p40 may have a role in the pathogenesis of the disease. The present study was undertaken to explore the role of p40 in the expression of TNF‐α in microglia. Interestingly, we have found that IL‐12 p70, p402 (the p40 homodimer) and p40 (the p40 monomer) dose‐dependently induced the production of TNF‐α in BV‐2 microglial cells. This induction of TNF‐α production was accompanied by an induction of TNF‐α mRNA. In addition to BV‐2 glial cells, p70, p402 and p40 also induced the production of TNF‐α in mouse primary microglia and peritoneal macrophages. Since the activation of both NF‐κB and C/EBPb is important for the expression of TNF‐α in microglial cells, we investigated the effect of p40 on the activation of NF‐κB as well as C/EBPb. Activation of NF‐κB as well as C/EBPb by p40 and inhibition of p40‐induced expression of TNF‐α by Dp65, a dominant‐negative mutant of p65, and DC/EBPb, a dominant‐negative mutant of C/EBPb, suggests that p40 induces the expression of TNF‐α through the activation of NF‐κB and C/EBPb. This study delineates a novel role of IL‐12 p40 in inducing the expression of TNF‐α in microglial cells which may participate in the pathogenesis of neuroinflammatory diseases. Acknowledgements: This study was supported by NIH grants (NS39940 and AG19487).     

NF‐κB activation in astrocytes drives a stage‐specific beneficial neuroimmunological response in ALS

Astrocytes are involved in non‐cell‐autonomous pathogenic cascades in amyotrophic lateral sclerosis (ALS); however, their role is still debated. We show that astrocytic NF‐κB activation drives microglial proliferation and leukocyte infiltration in the SOD1 (G93A) ALS model. This response prolongs the presymptomatic phase, delaying muscle denervation and decreasing disease burden, but turns detrimental in the symptomatic phase, accelerating disease progression. The transition corresponds to a shift in the microglial phenotype showing two effects that can be dissociated by temporally controlling NF‐κB activation. While NF‐κB activation in astrocytes induced a Wnt‐dependent microglial proliferation in the presymptomatic phase with neuroprotective effects on motoneurons, in later stage, astrocyte NF‐κB‐dependent microglial activation caused an accelerated disease progression. Notably, suppression of the early microglial response by CB₂R agonists had acute detrimental effects. These data identify astrocytes as important regulators of microglia expansion and immune response. Therefore, stage‐dependent microglia modulation may be an effective therapeutic strategy in ALS.     

Quantitative analysis of cellular proteome alterations in human influenza A virus‐infected mammalian cell lines

Over the last years virus–host cell interactions were investigated in numerous studies. Viral strategies for evasion of innate immune response, inhibition of cellular protein synthesis and permission of viral RNA and protein production were disclosed. With quantitative proteome technology, comprehensive studies concerning the impact of viruses on the cellular machinery of their host cells at protein level are possible. Therefore, 2‐D DIGE and nanoHPLC‐nanoESI‐MS/MS analysis were used to qualitatively and quantitatively determine the dynamic cellular proteome responses of two mammalian cell lines to human influenza A virus infection. A cell line used for vaccine production (MDCK) was compared with a human lung carcinoma cell line (A549) as a reference model. Analyzing 2‐D gels of the proteomes of uninfected and influenza‐infected host cells, 16 quantitatively altered protein spots (at least ±1.7‐fold change in relative abundance, p<0.001) were identified for both cell lines. Most significant changes were found for keratins, major components of the cytoskeleton system, and for Mx proteins, interferon‐induced key components of the host cell defense. Time series analysis of infection processes allowed the identification of further proteins that are described to be involved in protein synthesis, signal transduction and apoptosis events. Most likely, these proteins are required for supporting functions during influenza viral life cycle or host cell stress response. Quantitative proteome‐wide profiling of virus infection can provide insights into complexity and dynamics of virus–host cell interactions and may accelerate antiviral research and support optimization of vaccine manufacturing processes.     

Systematic Evaluation of Immobilized Trypsin‐Based Fast Protein Digestion for Deep and High‐Throughput Bottom‐Up Proteomics

Immobilized trypsin (IM) has been recognized as an alternative to free trypsin (FT) for accelerating protein digestion 30 years ago. However, some questions of IM still need to be answered. How does the solid matrix of IM influence its preference for protein cleavage and how well can IM perform for deep bottom‐up proteomics compared to FT? By analyzing Escherichia coli proteome samples digested with amine or carboxyl functionalized magnetic bead–based IM (IM‐N or IM‐C) or FT, it is observed that IM‐N with the nearly neutral solid matrix, IM‐C with the negatively charged solid matrix, and FT have similar cleavage preference considering the microenvironment surrounding the cleavage sites. IM‐N (15 min) and FT (12 h) both approach 9000 protein identifications (IDs) from a mouse brain proteome. Compared to FT, IM‐N has no bias in the digestion of proteins that are involved in various biological processes, are located in different components of cells, have diverse functions, and are expressed in varying abundance. A high‐throughput bottom‐up proteomics workflow comprising IM‐N‐based rapid protein cleavage and fast CZE‐MS/MS enables the completion of protein sample preparation, CZE‐MS/MS analysis, and data analysis in only 3 h, resulting in 1000 protein IDs from the mouse brain proteome.     

In‐depth characterization of the tomato fruit pericarp proteome

Since the genome of Solanum lycopersicum L. was published in 2012, some studies have explored its proteome although with a limited depth. In this work, we present an extended characterization of the proteome of the tomato pericarp at its ripe red stage. Fractionation of tryptic peptides generated from pericarp proteins by off‐line high‐pH reverse‐phase phase chromatography in combination with LC‐MS/MS analysis on a Fisher Scientific Q Exactive and a Sciex Triple‐TOF 6600 resulted in the identification of 8588 proteins with a 1% FDR both at the peptide and protein levels. Proteins were mapped through GO and KEGG databases and a large number of the identified proteins were associated with cytoplasmic organelles and metabolic pathways categories. These results constitute one of the most extensive proteome datasets of tomato so far and provide an experimental confirmation of the existence of a high number of theoretically predicted proteins. All MS data are available in the ProteomeXchange repository with the dataset identifiers PXD004947 and PXD004932.     

Proteomic analysis of rat microglia establishes a high-confidence reference data set of over 3000 proteins

Highly aggressively proliferating immortalized (HAPI) microglial cells have been used as an in vitro model for investigating key microglial functions including inflammatory, neurotoxic, and phagocytic activities. Through the use of offline strong cation-exchange fractionation followed by inline reversed-phase chromatographic separation and tandem mass spectrometric analysis on a hybrid linear ion trap-Orbitrap instrument, the HAPI microglial proteome was characterized to a depth of 3006 unique protein groups. Upon bioinformatic analysis of the HAPI proteome data set, enrichment was observed for processes relevant to microglial function including those associated with immune system response. This study marks the most comprehensive reference data set generated to date for the rat microglial proteome.     

Helicobacter pylori proteomics by 2‐DE/MS, 1‐DE‐LC/MS and functional data mining

With its predicted proteome of 1550 proteins (data set Etalon) Helicobacter pylori 26695 represents a perfect model system of medium complexity for investigating basic questions in proteomics. We analyzed urea‐solubilized proteins by 2‐DE/MS (data set 2‐DE) and by 1‐DE‐LC/MS (Supprot); proteins insoluble in 9 M urea but solubilized by SDS (Pellet); proteins precipitating in the Sephadex layer at the application side of IEF (Sephadex) by 1‐DE‐LC/MS; and proteins precipitating close to the application side within the IEF gel by LC/MS (Startline). The experimental proteomics data of H. pylori comprising 567 proteins (protein coverage: 36.6%) were stored in the Proteome Database System for Microbial Research ( http://www.mpiib‐berlin.mpg.de/2D‐PAGE/ ), which gives access to raw mass spectra (MALDI‐TOF/TOF) in T2D format, as well as to text files of peak lists. For data mining the protein mapping and comparison tool PROMPT ( http://webclu.bio.wzw.tum.de/prompt/ ) was used. The percentage of proteins with transmembrane regions, relative to all proteins detected, was 0, 0.2, 0, 0.5, 3.8 and 6.3% for 2‐DE, Supprot, Startline, Sephadex, Pellet, and Etalon, respectively. 2‐DE does not separate membrane proteins because they are insoluble in 9 M urea/70 mM DTT and 2% CHAPS. SDS solubilizes a considerable portion of the urea‐insoluble proteins and makes them accessible for separation by SDS‐PAGE and LC. The 2‐DE/MS analysis with urea‐solubilized proteins and the 1‐DE‐LC/MS analysis with the urea‐insoluble protein fraction (Pellet) are complementary procedures in the pursuit of a complete proteome analysis. Access to the PROMPT‐generated diagrams in the Proteome Database allows the mining of experimental data with respect to other functional aspects.     

Folic acid deficiency enhanced microglial immune response via the Notch1/nuclear factor kappa B p65 pathway in hippocampus following rat brain I/R injury and BV2 cells

Recent studies revealed that folic acid deficiency (FD) increased the likelihood of stroke and aggravated brain injury after focal cerebral ischaemia. The microglia‐mediated inflammatory response plays a crucial role in the complicated pathologies that lead to ischaemic brain injury. However, whether FD is involved in the activation of microglia and the neuroinflammation after experimental stroke and the underlying mechanism is still unclear. The aim of the present study was to assess whether FD modulates the Notch1/nuclear factor kappa B (NF‐κB) pathway and enhances microglial immune response in a rat middle cerebral artery occlusion‐reperfusion (MCAO) model and oxygen‐glucose deprivation (OGD)‐treated BV‐2 cells. Our results exhibited that FD worsened neuronal cell death and exaggerated microglia activation in the hippocampal CA1, CA3 and Dentate gyrus (DG) subregions after cerebral ischaemia/reperfusion. The hippocampal CA1 region was more sensitive to ischaemic injury and FD treatment. The protein expressions of proinflammatory cytokines such as tumour necrosis factor‐α, interleukin‐1β and interleukin‐6 were also augmented by FD treatment in microglial cells of the post‐ischaemic hippocampus and in vitro OGD‐stressed microglia model. Moreover, FD not only dramatically enhanced the protein expression levels of Notch1 and NF‐κB p65 but also promoted the phosphorylation of pIkBα and the nuclear translocation of NF‐κB p65. Blocking of Notch1 with N‐[N‐(3, 5‐difluorophenacetyl)‐l‐alanyl]‐S‐phenylglycine t‐butyl ester partly attenuated the nuclear translocation of NF‐κB p65 and the protein expression of neuroinflammatory cytokines in FD‐treated hypoxic BV‐2 microglia. These results suggested that Notch1/NF‐κB p65 pathway‐mediated microglial immune response may be a molecular mechanism underlying cerebral ischaemia‐reperfusion injury worsened by FD treatment.     

Proteomic analysis of cells in the early stages of herpes simplex virus type‐1 infection reveals widespread changes in the host cell proteome

During infection by herpes simplex virus type‐1 (HSV‐1) the host cell undergoes widespread changes in gene expression and morphology in response to viral replication and release. However, relatively little is known about the specific proteome changes that occur during the early stages of HSV‐1 replication prior to the global damaging effects of virion maturation and egress. To investigate pathways that may be activated or utilised during the early stages of HSV‐1 replication, 2‐DE and LC‐MS/MS were used to identify cellular proteome changes at 6 h post infection. Comparative analysis of multiple gels representing whole cell extracts from mock‐ and HSV‐1‐infected HEp‐2 cells revealed a total of 103 protein spot changes. Of these, 63 were up‐regulated and 40 down‐regulated in response to infection. Changes in selected candidate proteins were verified by Western blot analysis and their respective cellular localisations analysed by confocal microscopy. We have identified differential regulation and modification of proteins with key roles in diverse cellular pathways, including DNA replication, chromatin remodelling, mRNA stability and the ER stress response. This work represents the first global comparative analysis of HSV‐1 infected cells and provides an important insight into host cell proteome changes during the early stages of HSV‐1 infection.     

Rapid selection of peptide containing fractions in off‐line 2‐D HPLC in shotgun proteome analysis by screening with MALDI TOF MS

A simple and fast screening method for the selection of fractions of first dimension separation to be analyzed in second dimension‐MS/MS experiments in offline multidimensional liquid chromatographic separation schemes for shotgun proteome analysis was developed. The method is based on the measurement of total peptide content of the first dimension fractions by MALDI MS and was established using a tryptic digest of a bacterial proteome. The results of the screening process were in good agreement with those obtained in a detailed proteome analysis performed by RP×ion‐pair RP‐MALDI TOF/TOF MS analysis. The method supports a straightforward planning of experiments, also enabling a reduction of overall measurement time in shotgun proteome analysis.     

Adenosine‐to‐Inosine RNA Editing in Mouse and Human Brain Proteomes

Proteogenomics is based on the use of customized genome or RNA sequencing databases for interrogation of shotgun proteomics data in search for proteome‐level evidence of genome variations or RNA editing. In this work, the products of adenosine‐to‐inosine RNA editing in human and murine brain proteomes are identified using publicly available brain proteome LC‐MS/MS datasets and an RNA editome database compiled from several sources. After filtering of false‐positive results, 20 and 37 sites of editing in proteins belonging to 14 and 32 genes are identified for murine and human brain proteomes, respectively. Eight sites of editing identified with high spectral counts overlapped between human and mouse brain samples. Some of these sites have been previously reported using orthogonal methods, such as α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) glutamate receptors, CYFIP2, coatomer alpha. Also, differential editing between neurons and microglia is demonstrated in this work for some of the proteins from primary murine brain cell cultures. Because many edited sites are still not characterized functionally at the protein level, the results provide a necessary background for their further analysis in normal and diseased cells and tissues using targeted proteomic approaches.     

Characterisation of the nuclear proteome of a dehydration‐sensitive cultivar of chickpea and comparative proteomic analysis with a tolerant cultivar

Water deficit or dehydration hampers plant growth and development, and shrinks harvest size of major crop species worldwide. Therefore, a better understanding of dehydration response is the key to decipher the regulatory mechanism of better adaptation. In recent years, nuclear proteomics has become an attractive area of research, particularly to study the role of nucleus in stress response. In this study, a proteome of dehydration‐sensitive chickpea cultivar (ICCV‐2) was generated from nuclei‐enriched fractions. The LC‐MS/MS analysis led to the identification of 75 differentially expressed proteins presumably associated with different metabolic and regulatory pathways. Nuclear localisation of three candidate proteins was validated by transient expression assay. The ICCV‐2 proteome was then compared with that of JG‐62, a tolerant cultivar. The differential proteomics and in silico analysis revealed cultivar‐specific differential expression of many proteins involved in various cellular functions. The differential tolerance could be attributed to altered expression of many structural proteins and the proteins involved in stress adaptation, notably the ROS catabolising enzymes. Further, a comprehensive comparison on the abiotic stress‐responsive nuclear proteome was performed using the datasets published thus far. These findings might expedite the functional determination of the dehydration‐responsive proteins and their prioritisation as potential molecular targets for better adaptation.     

2‐DE proteome maps for the leaf apoplast of Nicotiana benthamiana

We provide 2‐D gel reference maps for the apoplastic proteome of Nicotiana benthamiana leaves infiltrated or not with the bacterial gene vector Agrobacterium tumefaciens. About 90 proteins were analyzed by LC‐MS/MS for identification and function assignment. We show, overall, an effective response of the plant to agroinfiltration involving a specific, cell wall maintenance‐independent up‐regulation of defense protein secretion. The proteome maps described should be a useful tool for systemic studies on plant–pathogen interactions or cell wall metabolism. They also should prove useful for the monitoring of secreted recombinant proteins and their possible pleiotropic effects along the cell secretory pathway of N. benthamiana leaves used as an expression platform for clinically useful proteins.     

Global analysis of the rat and human platelet proteome – the molecular blueprint for illustrating multi‐functional platelets and cross‐species function evolution

Emerging evidences indicate that blood platelets function in multiple biological processes including immune response, bone metastasis and liver regeneration in addition to their known roles in hemostasis and thrombosis. Global elucidation of platelet proteome will provide the molecular base of these platelet functions. Here, we set up a high‐throughput platform for maximum exploration of the rat/human platelet proteome using integrated proteomic technologies, and then applied to identify the largest number of the proteins expressed in both rat and human platelets. After stringent statistical filtration, a total of 837 unique proteins matched with at least two unique peptides were precisely identified, making it the first comprehensive protein database so far for rat platelets. Meanwhile, quantitative analyses of the thrombin‐stimulated platelets offered great insights into the biological functions of platelet proteins and therefore confirmed our global profiling data. A comparative proteomic analysis between rat and human platelets was also conducted, which revealed not only a significant similarity, but also an across‐species evolutionary link that the orthologous proteins representing “core proteome”, and the “evolutionary proteome” is actually a relatively static proteome.