Characterization of individual mouse cerebrospinal fluid proteomes

Analysis of cerebrospinal fluid (CSF) offers key insight into the status of the CNS. Characterization of murine CSF proteomes can provide a valuable resource for studying CNS injury and disease in animal models. However, the small volume of CSF in mice has thus far limited individual mouse proteome characterization. Through nonterminal CSF extractions in C57Bl/6 mice and high‐resolution 2D‐LC MS/MS analysis of individual murine samples, we report the most comprehensive proteome characterization of individual murine CSF to date. We identified a total of 566 unique proteins, including 128 proteins from three individual CSF samples that have been previously identified in brain tissue. Our methods and analysis provide a mechanism for individual murine CSF proteome analysis. The data are available in the ProteomeXchange with identifier PXD000248 ( http://proteomecentral.proteomexchange.org/dataset/PXD000248 ).     

The human oligodendrocyte proteome

Myelination of the CNS is performed by oligodendrocytes (OLs), which have been implicated in brain disorders, such as multiple sclerosis and schizophrenia. We have used the human oligodendroglial cell line MO3.13 to establish an OL reference proteome database. Proteins were prefractionationated by SDS‐PAGE and after in‐gel digestion subjected to nanoflow LC‐MS analysis. Approximately 11 600 unique peptides were identified and, after stringent filtering, resulted in 2290 proteins representing nine distinct biological processes and various molecular classes and functions. OL‐specific proteins, such as myelin basic protein (MBP) and 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP), as well as other proteins involved in multiple sclerosis and schizophrenia were also identified and are discussed. Proteins of this dataset have also been classified according to their chromosomal origin for providing useful data to the Chromosome‐centric Human Proteome Project (C‐HPP). Given the importance of OLs in the etiology of demyelinating and oligodendrogial disorders, the MO3.13 proteome database is a valuable data resource. The MS proteomics data have been deposited to the ProteomeXchange with identifier PXD000263 ( http://proteomecentral.proteomexchange.org/dataset/PXD000263 ).     

Qualitative and quantitative analysis of the adult Drosophila melanogaster proteome

Drosophila melanogaster is one of the most widely used model organisms in life sciences. Mapping its proteome is of great significance for understanding the biological characteristics and tissue functions of this species. However, the comprehensive coverage of its proteome remains a challenge. Here, we describe a high‐coverage analysis of whole fly through a 1D gel electrophoresis and LC‐MS/MS approach. By combining the datasets of two types of SDS‐PAGE and two kinds of tagmata, the high‐coverage analysis resulted in the identification of 5262 genes, which correspond to 38.23% of the entire coding genes. Moreover, we found that the fly head and body have different molecular weight distributions of their proteomes when the proteins were resolved with SDS‐PAGE and image analysis of the stained gel. This phenomenon was further confirmed by both label‐free and isobaric tags for relative and absolute quantitation‐based quantitative approaches. The consistent results of the two different quantitation methods also demonstrated the stability and accuracy of the LC‐MS/MS platform. The MS proteomics data have been deposited to the ProteomeXchange with identifiers PXD000454 and PXD000455 ( http://proteomecentral.proteomexchange.org/dataset/PXD000454 ; ( http://proteomecentral.proteomexchange.org/dataset/PXD000455 ).     

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.     

Gel‐free mass spectrometry analysis of Drosophila melanogaster heads

Membrane proteins play key roles in several fundamental biological processes such as cell signalling, energy metabolism and transport. Despite the significance, these still remain an under‐represented group in proteomics datasets. Herein, a bottom‐up approach to analyse an enriched membrane fraction from Drosophila melanogaster heads using multidimensional liquid chromatography (LC) coupled with tandem‐mass spectrometry (MS/MS) that relies on complete solubilisation and digestion of proteins, is reported. An enriched membrane fraction was prepared using equilibrium density centrifugation on a discontinuous sucrose gradient, followed by solubilisation using the filter‐aided sample preparation (FASP), tryptic and sequential chymotryptic digestion of proteins. Peptides were separated by reversed‐phase (RP) LC at high pH in the first dimension and acidic RP‐LC in the second dimension coupled directly to an Orbitrap Velos Pro mass spectrometer. A total number of 4812 proteins from 114 865 redundant and 38 179 distinct peptides corresponding to 4559 genes were identified in the enriched membrane fraction from fly heads. These included brain receptors, transporters and channels that are most important elements as drug targets or are linked to disease. Data are available via ProteomeXchange with identifier PXD001712 ( http://proteomecentral.proteomexchange.org/dataset/PXD001712 ).     

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 ).     

In‐depth characterization of the secretome of mouse CNS cell lines by LC‐MS/MS without prefractionation

Microglia, astrocytes, and neurons, which have important functions in the central nervous system (CNS), communicate mutually to generate a signal through secreted proteins or small molecules, but many of which have not been identified. Because establishing a reference for the secreted proteins from CNS cells could be invaluable in examining cell‐to‐cell communication in the brain, we analyzed the secretome of three murine CNS cell lines without prefractionation by high‐resolution mass spectrometry. In this study, 2795 proteins were identified from conditioned media of the three cell lines, and 2125 proteins were annotated as secreted proteins by bioinformatics analysis. Further, approximately 500 secreted proteins were quantifiable as differentially expressed proteins by label‐free quantitation. As a result, our secretome references are useful datasets for the future study of neuronal diseases. All MS data have been deposited in the ProteomeXchange with identifier PXD001597 ( http://proteomecentral.proteomexchange.org/dataset/PXD001597 ).     

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.     

The lipid raft proteome of Borrelia burgdorferi

Eukaryotic lipid rafts are membrane microdomains that have significant amounts of cholesterol and a selective set of proteins that have been associated with multiple biological functions. The Lyme disease agent, Borrelia burgdorferi, is one of an increasing number of bacterial pathogens that incorporates cholesterol onto its membrane, and form cholesterol glycolipid domains that possess all the hallmarks of eukaryotic lipid rafts. In this study, we isolated lipid rafts from cultured B. burgdorferi as a detergent resistant membrane (DRM) fraction on density gradients, and characterized those molecules that partitioned exclusively or are highly enriched in these domains. Cholesterol glycolipids, the previously known raft‐associated lipoproteins OspA and OpsB, and cholera toxin partitioned into the lipid rafts fraction indicating compatibility with components of the DRM. The proteome of lipid rafts was analyzed by a combination of LC‐MS/MS or MudPIT. Identified proteins were analyzed in silico for parameters that included localization, isoelectric point, molecular mass and biological function. The proteome provided a consistent pattern of lipoproteins, proteases and their substrates, sensing molecules and prokaryotic homologs of eukaryotic lipid rafts. This study provides the first analysis of a prokaryotic lipid raft and has relevance for the biology of Borrelia, other pathogenic bacteria, as well as for the evolution of these structures. All MS data have been deposited in the ProteomeXchange with identifier PXD002365 ( http://proteomecentral.proteomexchange.org/dataset/PXD002365 ).     

Differential Ultrastructural Localization of Myelin Basic Protein, Myelin/Oligodendroglial Glycoprotein, and 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase in the CNS of Adult Rats

In a light and electron microscopic immunocytochemical study we have examined the distribution of myelin basic protein (MBP), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), and myelin/oligodendroglial glycoprotein (MOG) within CNS myelin sheaths and oligodendrocytes of adult Sprague-Dawley rats. Ultrastructural immunocytochemistry allowed quantitative analysis of antigen density in different myelin and oligodendrocyte zones: MBP was detectable in high density over the whole myelin sheath, but not in regions of loops, somata, or the oligodendrocyte plasma membrane. CNP reactivity was highest at the myelin/axon interface, and found in lower concentration over the outer lamellae of myelin sheaths, at the cytoplasmic face of oligodendrocyte membranes, and throughout the compact myelin. MOG was preferentially detected at the extracellular surface of myelin sheaths and oligodendrocytes and in only low amounts in the lamellae of compacted myelin and the myelin/axon border zone. Our studies, thus, indicate further the presence of different molecular domains in compact myelin, which may be functionally relevant for the integrity and maintenance of the myelin sheath.     

Proteomic and phosphoproteomic analyses of yeast reveal the global cellular response to sphingolipid depletion

Sphingolipids are essential components of eukaryotic cells with important functions in membrane biology and cellular signaling. Their levels are tightly controlled and coordinated with the abundance of other membrane lipids. How sphingolipid homeostasis is achieved is not yet well understood. Studies performed primarily in yeast showed that the phosphorylation states of several enzymes and regulators of sphingolipid synthesis are important, although a global understanding for such regulation is lacking. Here, we used high‐resolution MS‐based proteomics and phosphoproteomics to analyze the cellular response to sphingolipid synthesis inhibition. Our dataset reveals that changes in protein phosphorylation, rather than protein abundance, dominate the response to blocking sphingolipid synthesis. We identified Ypk signaling as a pathway likely to be activated under these conditions, and we identified potential Ypk1 target proteins. Our data provide a rich resource for on‐going mechanistic studies of key elements of the cellular response to the depletion of sphingolipid levels and the maintenance of sphingolipid homeostasis. All MS data have been deposited in the ProteomeXchange with identifier PXD003854 ( http://proteomecentral.proteomexchange.org/dataset/PXD003854 ).     

Updating the proteome of the uncultivable hemotrophic Mycoplasma suis in experimentally infected pigs

Mycoplasma suis belongs to the hemotrophic mycoplasmas that are associated with acute and chronic anemia in a wide range of livestock and wild animals. The inability to culture M. suis in vitro has hindered its characterization at the molecular level. Since the publication of M. suis genome sequences in 2011 only one proteome study has been published. Aim of the presented study was to significantly extend the proteome coverage of M. suis strain KI_3806 during acute infection by applying three different protein extraction methods followed by 1D SDS‐PAGE and LC‐MS/MS. A total of 404 of 795 M. suis KI_3806 proteins (50.8%) were identified. Data analysis revealed the expression of 83.7% of the predicted ORFs with assigned functions but also highlights the expression of 179 of 523 (34.2%) hypothetical proteins with unknown functions. Computational analyses identified expressed membrane‐associated hypothetical proteins that might be involved in adhesion or host–pathogen interaction. Furthermore, analyses of the expressed proteins indicated the existence of a hexose‐6‐phosphate‐transporter and an ECF transporter. In conclusion, our proteome study provides a further step toward the elucidation of the unique life cycle of M. suis and the establishment of an in vitro culture. All MS data have been deposited in the ProteomeXchange with identifier PXD002294 ( http://proteomecentral.proteomexchange.org/dataset/PXD002294 ).     

In‐depth protein profiling of the postsynaptic density from mouse hippocampus using data‐independent acquisition proteomics

Located at neuronal terminals, the postsynaptic density (PSD) is a highly complex network of cytoskeletal scaffolding and signaling proteins responsible for the transduction and modulation of glutamatergic signaling between neurons. Using ion‐mobility enhanced data‐independent label‐free LC‐MS/MS, we established a reference proteome of crude synaptosomes, synaptic junctions, and PSD derived from mouse hippocampus including TOP3‐based absolute quantification values for identified proteins. The final dataset across all fractions comprised 49 491 peptides corresponding to 4558 protein groups. Of these, 2102 protein groups were identified in highly purified PSD in at least two biological replicates. Identified proteins play pivotal roles in neurological and synaptic processes providing a rich resource for studies on hippocampal PSD function as well as on the pathogenesis of neuropsychiatric disorders. All MS data have been deposited in the ProteomeXchange with identifier PXD000590 ( http://proteomecentral.proteomexchange.org/dataset/PXD000590 ).     

Proteomic snapshot of spearmint (Mentha spicata L.) leaf trichomes: A genuine terpenoid factory

Peltate glandular trichomes from Mentha spicata were purified on a Percoll gradient and soluble and membrane proteins were trypsinized and the peptides were separated by nano‐LC fractionation and analyzed by MALDI‐MS/MS. The vast majority of the 1666 proteins identified were housekeeping proteins or involved in the primary metabolism. However, 57 were predicted to be involved in the secondary metabolism. Of these, 21 were involved in the synthesis of phenylpropanoids and phenolics and 32 in terpenoid synthesis. Of the 14 membrane transporters identified, the 11 ATP‐binding cassette transporters provide good material for assessing whether active transport is required for the transfer of monoterpenoid intermediates between cellular compartments and for the secretion of the final products into the subcuticular storage cavity. In conclusion, this proteome analysis of M. spicata peltate trichomes has identified several candidate proteins that might be involved in terpenoid synthesis and transport. The data have been deposited to the ProteomeXchange with identifier PXD000352 ( http://proteomecentral.proteomexchange.org/dataset/PXD000352 ).     

Proteomic identification of rainbow trout seminal plasma proteins

In the study, the combination of protein fractionation by 1DE and HPLC‐ESI‐MS/MS was used to characterize the rainbow trout seminal plasma proteome. Our results led to the creation of a catalogue of rainbow trout seminal plasma proteins (152 proteins) and significantly contributed to the current knowledge regarding the protein composition of fish seminal plasma. The major proteins of rainbow trout seminal plasma, such as transferrin, apolipoproteins, complement C3, serum albumin, and hemopexin‐, alpha‐1‐antiproteinase‐, and precerebellin‐like protein, were recognized as acute‐phase proteins (proteins that plasma concentration changes in response to inflammation). This study provides the basis for further functional studies of fish seminal plasma proteins, as well as for the identification of novel biomarkers for sperm quality. The MS data have been deposited in the ProteomeXchange with identifier PXD000306 ( http://proteomecentral.proteomexchange.org/dataset/PXD000306 ).     

Differential quantitative proteome analysis of Escherichia coli grown on acetate versus glucose

Relative protein abundances of Escherichia coli MG1655 growing exponentially on minimal medium with acetate or glucose as the sole carbon source were investigated in a quantitative shotgun proteome analysis with TMT6‐plex isobaric tags. Peptides were separated by high resolution high/low pH 2D‐LC, using an optimized fraction pooling scheme followed by mass spectrometric analysis. Quantitative data were acquired for 2099 proteins covering 49% of the predicted E. coli proteins, showing system‐wide effects of growth conditions. In total, 507 proteins showed a fold change of at least 1.5 and 205 proteins changed by more than twofold. Significant differences in abundance were observed for most of the proteins in the central carbon metabolism and in proteins relevant for amino acid and protein synthesis, processing of environmental information and scavenging of a variety of alternate carbon sources. Periplasmic‐binding proteins were also more abundant on acetate, especially proteins involved in scavenging extracellular resources such as sugars. All MS data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (dataset identifier PXD003863).     

Loss of neprilysin alters protein expression in the brain of Alzheimer's disease model mice

Alzheimer's disease (AD) is a neurodegenerative disease displaying extracellular plaques formed by the neurotoxic amyloid β‐peptide (Aβ), and intracellular neurofibrillary tangles consisting of protein tau. However, how these pathologies relate to the massive neuronal death that occurs in AD brains remain elusive. Neprilysin is the major Aβ‐degrading enzyme and a lack thereof increases Aβ levels in the brain twofold. To identify altered protein expression levels induced by increased Aβ levels, we performed a proteomic analysis of the brain of the AD mouse model APPsw and compared it to that of APPsw mice lacking neprilysin. To this end we established an LC‐MS/MS method to analyze brain homogenate, using an ¹⁸O‐labeled internal standard to accurately quantify the protein levels. To distinguish between alterations in protein levels caused by increased Aβ levels and those induced by neprilysin deficiency independently of Aβ, the brain proteome of neprilysin deficient APPsw mice was also compared to that of neprilysin deficient mice. By this approach we identified approximately 600 proteins and the levels of 300 of these were quantified. Pathway analysis showed that many of the proteins with altered expression were involved in neurological disorders, and that tau, presenilin and APP were key regulators in the identified networks. The data have been deposited to the ProteomeXchange Consortium with identifiers PXD000968 and PXD001786 ( http://proteomecentral.proteomexchange.org/dataset/PXD000968 and ( http://proteomecentral.proteomexchange.org/dataset/PXD001786 ). Interestingly, the levels of several proteins, including some not previously reported to be linked to AD, were associated with increased Aβ levels.