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 ).     

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 ).     

Reference proteome of highly purified human Th1 cells reveals strong effects on metabolism and protein ubiquitination upon differentiation

The differentiation of human CD4⁺ T cells into T helper cell subtypes and regulatory T cells is crucial to the immune response. Among subtypes, Th1 cells are dominant, representing approximately 50% of all lymphocytes. Thus far, most global proteomic studies have used only partially purified T helper cell subpopulations and/or have employed artificial protocols for inducing specific T helper cell subtypes and/or used gel‐based approaches. These studies have shed light on molecular details of certain aspects of the proteome; nevertheless a global analysis of high purity primary naïve and Th1 cells by LC‐MS/MS is required to provide a reference dataset for proteome‐based T cell subtype characterization. The utilization of highly purified Th1 cells for a global proteome assessment and the bioinformatic comparison to naïve cells reveals changes in cell metabolism and the ubiquitination pathway upon T cell differentiation. All MS data have been deposited in the ProteomeXchange with identifier PXD001066 ( http://proteomecentral.proteomexchange.org/dataset/PXD001066 ).     

Proteomic analysis of outer membrane vesicles from the probiotic strain Escherichia coli Nissle 1917

Escherichia coli Nissle 1917 (EcN) is a probiotic used for the treatment of intestinal disorders. EcN improves gastrointestinal homeostasis and microbiota balance; however, little is known about how this probiotic delivers effector molecules to the host. Outer membrane vesicles (OMVs) are constitutively produced by Gram‐negative bacteria and have a relevant role in bacteria–host interactions. Using 1D SDS–PAGE and highly sensitive LC–MS/MS analysis we identified in this study 192 EcN vesicular proteins with high confidence in three independent biological replicates. Of these proteins, 18 were encoded by strain‐linked genes and 57 were common to pathogen‐derived OMVs. These proteins may contribute to the ability of this probiotic to colonize the human gut as they fulfil functions related to adhesion, immune modulation or bacterial survival in host niches. This study describes the first global OMV proteome of a probiotic strain and provides evidence that probiotic‐derived OMVs contain proteins that can target these vesicles to the host and mediate their beneficial effects on intestinal function. All MS data have been deposited in the ProteomeXchange with identifier PXD000367 ( http://proteomecentral.proteomexchange.org/dataset/PXD000367 ).     

Protein identification and quantification from riverbank grape,Vitis riparia: Comparing SDS‐PAGE and FASP‐GPF techniques for shotgun proteomic analysis

Protein sample preparation optimisation is critical for establishing reproducible high throughput proteomic analysis. In this study, two different fractionation sample preparation techniques (in‐gel digestion and in‐solution digestion) for shotgun proteomics were used to quantitatively compare proteins identified in Vitis riparia leaf samples. The total number of proteins and peptides identified were compared between filter aided sample preparation (FASP) coupled with gas phase fractionation (GPF) and SDS‐PAGE methods. There was a 24% increase in the total number of reproducibly identified proteins when FASP‐GPF was used. FASP‐GPF is more reproducible, less expensive and a better method than SDS‐PAGE for shotgun proteomics of grapevine samples as it significantly increases protein identification across biological replicates. Total peptide and protein information from the two fractionation techniques is available in PRIDE with the identifier PXD001399 ( http://proteomecentral.proteomexchange.org/dataset/PXD001399 ).     

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 ).     

Protein 2DE reference map of the anterior midgut of the blood‐sucking bug Rhodnius prolixus

Rhodnius prolixus is an important vector of Trypanosoma cruzi, the causative agent of Chagas’ disease, an illness that affects 20% of Latin America population. The obligatory course of the parasite in the vector digestive tract has made it an important target for investigation in order to control the parasite transmission and thus interrupt its biological cycle in the insect vector. Therefore, an insight into the vector midgut physiology is valuable for insect control as well as to provide potential novel targets for drugs and vaccines development and thus disease treatment. In this study, the first 2DE map of R. prolixus anterior midgut is described. Proteins were separated by 2DE and analyzed by nano‐LC MS/MS. The results yielded 489 proteins from 475 spots. These proteins were classified into 28 functional groups and their physiological roles in the insect midgut are discussed. All MS data have been deposited in the ProteomeXchange with identifiers PXD001488 and PXD001489 ( http://proteomecentral.proteomexchange.org/dataset/PXD001488 , http://proteomecentral.proteomexchange.org/dataset/PXD001489 ).     

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 ).     

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.     

Identification of inositol 1,4,5‐trisphosphate‐binding proteins by heparin‐agarose affinity purification and LTQ ORBITRAP MS in Oryza sativa

Inositol 1,4,5‐trisphosohate (IP₃) and its receptors play a pivotal role in calcium signal transduction in mammals. However, no homologs of mammalian IP₃ receptors have been found in plants. In this study, we isolated the microsomal fractions from rice cells in suspension culture and further obtained putative IP₃‐binding proteins by heparin‐agarose affinity purification. The IP₃‐binding activities of these protein fractions were determined by [³H] IP₃‐binding assay. SDS‐PAGE and MS analysis were then performed to characterize these proteins. We have identified 297 proteins from the eluates of heparin‐agarose column chromatography, which will provide insight into the IP₃ signaling pathways in plants. All MS data have been deposited in the ProteomeXchange with identifier PXD000763 ( http://proteomecentral.proteomexchange.org/dataset/PXD000763 ).     

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 ).     

Distinct serum proteome profiles associated with collagen‐induced arthritis and complete Freund's adjuvant‐induced inflammation in CD38−/− mice: The discriminative power of protein species or proteoforms

Collagen‐type‐II‐induced arthritis (CIA) is an autoimmune disease, which involves a complex host systemic response including inflammatory and autoimmune reactions. CIA is milder in CD38^?/? than in wild‐type (WT) mice. ProteoMiner‐equalized serum samples were subjected to 2D‐DiGE and MS‐MALDI‐TOF/TOF analyses to identify proteins that changed in their relative abundances in CD38^?/? versus WT mice either with arthritis (CIA⁺), with no arthritis (CIA^?), or with inflammation (complete Freund's adjuvant (CFA)‐treated mice). Multivariate analyses revealed that a multiprotein signature (n = 28) was able to discriminate CIA⁺ from CIA^? mice, and WT from CD38^?/? mice within each condition. Likewise, a distinct multiprotein signature (n = 16) was identified which differentiated CIA⁺ CD38^?/? mice from CIA⁺ WT mice, and lastly, a third multiprotein signature (n = 18) indicated that CD38^?/? and WT mice could be segregated in response to CFA treatment. Further analyses showed that the discriminative power to distinguish these groups was reached at protein species level and not at the protein level. Hence, the need to identify and quantify proteins at protein species level to better correlate proteome changes with disease processes. It is crucial for plasma proteomics at the low‐abundance protein species level to apply the ProteoMiner enrichment. All MS data have been deposited in the ProteomeXchange with identifiers PXD001788, PXD001799 and PXD002071 ( http://proteomecentral.proteomexchange.org/dataset/PXD001788 , http://proteomecentral.proteomexchange.org/dataset/PXD001799 and http://proteomecentral.proteomexchange.org/dataset/PXD002071 ).     

Compartment resolved reference proteome map from highly purified naïve,activated, effector,and memory CD8+ murine immune cells

Differentiation of CD8⁺ T lymphocytes into effector and memory cells is key for an adequate immune response and relies on complex interplay of pathways that convey signals from the cell surface to the nucleus. In this study, we investigated the proteome of four cytotoxic T‐cell subtypes; naïve, recently activated effector, effector, and memory cells. Cells were fractionated into membrane, cytosol, soluble nuclear, chromatin‐bound, and cytoskeletal compartments. Following LC‐MS/MS analysis, identified peptides were analyzed via MaxQuant. Compartment fractionation and gel‐LC‐MS separation resulted in 2399 proteins identified in total. Comparison between the different subsets resulted in 146 significantly regulated proteins for naïve and effector cells, followed by 116 for activated, and 55 for memory cells. Besides Granzyme B signaling (for activated and/ or effector cells vs. naïve cells), the most prominent changes occurred in the TCA cycle and aspartate degradation. These changes suggest that correct balancing of metabolism is key for differentiation processes. All MS data have been deposited in the ProteomeXchange with identifier PXD001065 ( http://proteomecentral.proteomexchange.org/dataset/PXD001065 ).     

Proteomic analysis of extracellular vesicles derived from Mycobacterium tuberculosis

The release of extracellular vesicles, also known as outer membrane vesicles, membrane vesicles, exosomes, and microvesicles, is an evolutionarily conserved phenomenon from bacteria to eukaryotes. It has been reported that Mycobacterium tuberculosis releases extracellular vesicles harboring immunologically active molecules, and these extracellular vesicles have been suggested to be applicable in vaccine development and biomarker discovery. However, the comprehensive proteomic analysis has not been performed for M. tuberculosis extracellular vesicles. In this study, we identified a total of 287 vesicular proteins by four LC‐MS/MS analyses with high confidence. In addition, we identified several vesicular proteins associated with the virulence of M. tuberculosis. This comprehensive proteome profile will help elucidate the pathogenic mechanism of M. tuberculosis. The data have been deposited to the ProteomeXchange with identifier PXD001160 ( http://proteomecentral.proteomexchange.org/dataset/PXD001160 ).     

Proteome profile of the endomembrane of developing coleoptiles from switchgrass (Panicum virgatum)

The cost‐effective production of biofuels from lignocellulosic material will likely require manipulation of plant biomass, specifically cell walls. The North American native prairie grass Panicum virgatum (switchgrass) is seen as a potential biofuel crop with an array of genetic resources currently being developed. We have characterized the endomembrane proteome of switchgrass coleoptiles to provide additional information to the switchgrass community. In total, we identified 1750 unique proteins from two biological replicates. These data have been deposited in the ProteomeXchange with the identifier PXD001351 ( http://proteomecentral.proteomexchange.org/dataset/PXD001351 ).     

Proteomic analysis of the thermophilic methylotroph Bacillus methanolicus MGA3

Bacillus methanolicus MGA3 is a facultative methylotroph of industrial relevance that is able to grow on methanol as its sole source of carbon and energy. The Gram‐positive bacterium possesses a soluble NAD⁺‐dependent methanol dehydrogenase and assimilates formaldehyde via the ribulose monophosphate (RuMP) cycle. We used label‐free quantitative proteomics to generate reference proteome data for this bacterium and compared the proteome of B. methanolicus MGA3 on two different carbon sources (methanol and mannitol) as well as two different growth temperatures (50°C and 37°C). From a total of approximately 1200 different detected proteins, approximately 1000 of these were used for quantification. While the levels of 213 proteins were significantly different at the two growth temperatures tested, the levels of 109 proteins changed significantly when cells were grown on different carbon sources. The carbon source strongly affected the synthesis of enzymes related to carbon metabolism, and in particular, both dissimilatory and assimilatory RuMP cycle enzyme levels were elevated during growth on methanol compared to mannitol. Our data also indicate that B. methanolicus has a functional tricarboxylic acid cycle, the proteins of which are differentially regulated on mannitol and methanol. Other proteins presumed to be involved in growth on methanol were constitutively expressed under the different growth conditions. All MS data have been deposited in the ProteomeXchange with the identifiers PXD000637 and PXD000638 ( http://proteomecentral.proteomexchange.org/dataset/PXD000637 , http://proteomecentral.proteomexchange.org/dataset/PXD000638 ).     

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 ).