Prediction and evaluation of the effect of pre-centrifugation sample management on the measurable untargeted LC-MS plasma metabolome.

Optimal handling is the most important means to ensure adequate sample quality. We aimed to investigate whether pre-centrifugation delay time and temperature could be accurately predicted and to what extent variability induced by pre-centrifugation management can be adjusted for. We used untargeted liquid chromatography-mass spectrometry (LC-MS) metabolomics to predict and evaluate the influence of pre-centrifugation temperature and delayed time on plasma samples. Pre-centrifugation temperature (4, 25 and 37 °C; classification rate 87%) and time (5-210 min; Q2 = 0.82) were accurately predicted using Random Forest (RF). Metabolites uniquely reflecting temperature and temperature-time interactions were discovered using a combination of RF and generalized linear models. Time-related metabolite profiles suggested a perturbed stability of the metabolome at all temperatures in the investigated time period (5-210 min), and the variation at 4 °C was observed in particular before 90 min. Fourteen and eight metabolites were selected and validated for accurate prediction of pre-centrifugation temperature (classification rate 94%) and delay time (Q2 = 0.90), respectively. In summary, the metabolite profile was rapidly affected by pre-centrifugation delay at all temperatures and thus the pre-centrifugation delay should be as short as possible for metabolomics analysis. The metabolite panels provided accurate predictions of pre-centrifugation delay time and temperature in healthy individuals in a separate validation sample. Such predictions could potentially be useful for assessing legacy samples where relevant metadata is lacking. However, validation in larger populations and different phenotypes, including disease states, is needed.

The EuPA Biobank Initiative: Meeting the future challenges of biobanking in proteomics & systems medicine.

In this News & Reviews Discussion, the recently launched EuPA (European Proteomics Association) Biobank Initiative is introduced in the context of current and future challenges in biobanking. The purpose of the initiative is to provide a forumand knowledge platform for integrating the extensive experiences collected by the EuPA community, and link it to the European and international biobanking communities at large. The specific impact of providing a forum and easy access to this type of information to the EuPA community is the potential of improving the quality of future sample collections and biobanks, the quality of the research produced from these sample collections, as well as the output and productivity from existing biobanks. The underutilization of biobanks has recently been identified as an emerging issue of biobankingworldwide. Measures to improve our ability to locate and access appropriate sample collections for a wide range of research purposes may enhance both the scientific quality and biobank sustainability, thereby contributing to the important task of moving our research beyond basic findings and mere publications, into clinical practice. BIOLOGICAL SIGNIFICANCE: This manuscript is intended as a Discussion piece, and represents a recollection of the presentation under the "EuPA Initiative" session at HUPO/EuPA 2014 in Madrid. The launch of the EuPA (European Proteomics Association) Biobank Initiative in the context of current and future challenges in biobanking is discussed. The purpose of the initiative is to provide a forum and knowledge base for integrating the extensive experiences collected by the EuPA community, and link it to the European and international biobanking communities at large. The specific impact of providing a forumand easy access to this type of information to the EuPA community is the potential of improving the quality of future sample collections and biobanks, the quality of the research produced from these sample collections, aswell as the output and productivity fromexisting biobanks. The underutilization of biobanks has recently been identified as a challenge to the well-being and economic sustainability of biobanking worldwide. Measures to improve our ability to localize and access appropriate sample collections for validation studies and other research purposes is thus of benefit bothto scientific quality and biobank sustainability, thereby contributing to the important task of moving our research beyond basic findings and mere publications, into clinical practice. This article is part of a Special Issue entitled: HUPO 2014.

Expansion of multipotent stem cells from the adult human brain.

The discovery of stem cells in the adult human brain has revealed new possible scenarios for treatment of the sick or injured brain. Both clinical use of and preclinical research on human adult neural stem cells have, however, been seriously hampered by the fact that it has been impossible to passage these cells more than a very few times and with little expansion of cell numbers. Having explored a number of alternative culturing conditions we here present an efficient method for the establishment and propagation of human brain stem cells from whatever brain tissue samples we have tried. We describe virtually unlimited expansion of an authentic stem cell phenotype. Pluripotency proteins Sox2 and Oct4 are expressed without artificial induction. For the first time multipotency of adult human brain-derived stem cells is demonstrated beyond tissue boundaries. We characterize these cells in detail in vitro including microarray and proteomic approaches. Whilst clarification of these cells' behavior is ongoing, results so far portend well for the future repair of tissues by transplantation of an adult patient's own-derived stem cells.

Effect of levetiracetam on hippocampal protein expression and cell proliferation in rats.

PURPOSE: Levetiracetam (LEV) is a broad-spectrum antiepileptic drug (AED) with possibly also antiepileptogenic properties. LEV has a specific binding site in the central nervous system and reduces brain excitability; however, the precise mechanism of action (MOA) of LEV remains unclear. To further unravel the potential MOA pathways of LEV we investigated altered protein expression and cell proliferation in rat hippocampal tissue during LEV administration. METHODS: On day 1 of the experiment, rats were randomly assigned to a treatment group (LEV, 600 mg/kg/day, n=10) and a control group (saline, n=10). On days 2 and 3 rats were injected with bromodeoxyuridine (50 mg/kg, i.p., BID). After 7 days of treatment rats were killed and their brains removed. The right hemisphere was processed for histochemistry. The left hippocampus was dissected and frozen for proteomic analysis. Proteins were extracted from the tissue and two-dimensional gel electrophoresis was performed. RESULTS: Treatment with LEV did not influence hippocampal cell proliferation. Multivariate analysis of differential protein expression, determined by proteomic analysis, revealed a significant clustering of control and treatment groups. The proteins which most contribute to the difference between groups were identified with mass spectrometry. The identified proteins were either involved in cytoskeleton, energy metabolism, neurotransmission, signal transduction, myelinization or stress response. DISCUSSION: LEV does not affect hippocampal cell proliferation but has significant effects on the expression of proteins involved in a variety of physiological processes involved in physiological processes that play an important role in the currently identified MOAs of AEDs such as neurotransmission and signal transduction.

Defining primary and secondary progenitor disorders in the brain: proteomic approaches for analysis of neural progenitor cells.

Since the discovery of endogenous progenitor cells in two brain regions in the adult, the notion that progenitor cells might be useful for repairing damaged neurons or replacing dead neurons has gone from fiction to a reality, at least in the laboratory setting. Progenitor cells have the unique ability to be able to produce new neurons in response to endogenous and exogenous cues from their microenvironment in the brain and from the environment of the organism. However, in models of several disorders and insults the regenerative potential of the central nervous system need external enhancing. In this review we begin by focussing on the developments in the field of neurobiology that have led to the specific study of neural progenitor cell biology. In particular we discuss the two germinal niches, the subventricular zone and the subgranular zone, as well as how various neurological diseases affect these niches. We furthermore try to define primary progenitor cell disorders and secondary progenitor cell responses. The second part of this review focuses on proteomic approaches for studying progenitor cells. These techniques allow the array of proteins that are expressed by progenitor cells to be determined and further more allow comparisons between diseased and normal cells or treated and untreated cell populations. If we can induce neural progenitor cells to generate functional neurons in the central nervous system (CNS) then the burden of neurological disorders may be eased in the future. The advances in proteomic technology have and will enable further understanding of the regulatory processes in these cells so that progenitor cell integration and differentiation can be enhanced. Hopefully an increase in knowledge of progenitor cell biology will have a major impact on clinical practice.

Proteomics and peptidomics in neuroscience. Experience of capabilities and limitations in a neurochemical laboratory.

The increasing use of proteomics has created a basis for new strategies to develop methodologies for rapid identification of protein patterns in living organisms. It has also become evident that proteomics has other potential applications than protein and peptide identification, e.g. protein characterization, with the aim of revealing their structure, function(s) and interactions of proteins. In comparative proteomics studies, the protein expression of a certain biological system is compared with another system or the same system under perturbed conditions. Global identification of proteins in neuroscience is extremely complex, owing to the limited availability of biological material and very low concentrations of the molecules. Moreover, in addition to proteins, there are number of peptides that must also be considered in global studies on the central nervous system. In this overview, we focus on and discuss problems related to the different sources of biological material and sample handling, which are part of all preparatory and analytical steps. Straightforward protocols are desirable to avoid excessive purification steps, since loss of material at each step is inevitable. We would like to merge the two worlds of proteomics/peptidomics and neuroscience, and finally we consider different practical and technical aspects, illustrated with examples from our laboratory.

Effects on rat thalamic proteome by acute and subchronic MK-801-treatment.

Since the symptoms of intoxication with non-competitive N-methyl-D-aspartate (NMDA) receptor antagonists closely mimic symptoms in patients with schizophrenia, [+]-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]-cycloheptene-5,10-iminehydrogenmaleate (MK-801)-treated rodents are often used as a model for schizophrenia. In most studies, acute injections of MK-801 to rats have been used, but in some studies, longer periods of treatment have been performed. In our previous work, alterations in mRNA/protein expression were screened in the cerebral cortex of MK-801 treated rats. Different proteins were altered in different treatment courses of MK-801. The main objective of the present study was to evaluate different treatment periods of treatment with MK-801 in rats as a model for schizophrenia. Thalamus proteins from treated (acute, six and 12 days) and control rats were analyzed with two-dimensional gel electrophoresis and mass spectrometry. Our results show that different treatment times of MK-801 to rats give different biochemical results. Therefore, it is important to use the same treatment time in studies that will be compared.

Comparative proteome analysis of thalamus in MK-801-treated rats.

Two-dimensional gel-electrophoresis in combination with mass spectrometry is a powerful approach to compare protein expression in brain tissues. Using this proteomic approach, and based on the hypothesis that schizophrenia involves hypoglutamergic brain function, alterations in protein levels in the thalamus of rats treated with the N-methyl-D-aspartate (NMDA) receptor antagonist [+]-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]-cycloheptene-5,10-iminehydrogenmaleate (MK-801), as compared to saline-treated animals, were assessed in an unbiased fashion. The rats were divided into two groups; group 1 (short-term treated) and group 2 (long-term treated). In group 1, the levels of seven proteins were increased and four proteins reduced. In group 2, the levels of six proteins were reduced. Several of the altered proteins (heat shock proteins 60 and 72, albumin, dihydropyrimidinase related protein-2, aldolase c, and malate dehydrogenase) have previously been connected to schizophrenia. Alterations of other proteins (dihydrolipoamide acetyltransferase component of pyruvate dehydrogenase complex E2, guanine deaminase, alpha-enolase, aconitase, ATP-synthase and alpha-internexin), have not, to the best of our knowledge, earlier been implicated in schizophrenia pathology. Our results show the high potential of using proteomic methods for the validation of animal models of schizophrenia and to identify new proteins involved in the pathophysiological mechanisms of schizophrenia.

Comparative genome- and proteome analysis of cerebral cortex from MK-801-treated rats.

cDNA microarrays and two-dimensional gel-electrophoresis in combination with mass spectrometry, were used to screen alterations in mRNA and protein levels, respectively, in cerebral cortex of MK-801-treated rats. The rats were divided in two groups; group 1 (short-term treated) and group 2 (long-term treated). In group 1, four genes were up-regulated and five down-regulated. In group 2, seven genes were up-regulated and six down-regulated. In group 1, the levels of one protein was increased and eight proteins reduced. In group 2, the levels of two proteins were increased and four proteins reduced. Several of the altered genes (casein kinase 2, glutamic acid decarboxylase, synaptotagmin, gamma aminobutyric acid [GABA] transporter, creatine kinase, and cytochrome c oxidase) and proteins (superoxide dismutase, hsp 60, hsp 72 and gamma-enolase) have previously been connected to schizophrenia. Alterations of the genes (microglobulin, c-jun proto-oncogene, 40S ribosomal protein S19, adenosine diphosphate (ADP)-ribosylation factors, platelet-derived growth factor, fructose-bisphophate aldolase A, and myelin proteolipid) and the proteins (stathmin, H+-transp. Adenosine triphosphate (ATP) synthase, pyruvate dehydrogenase, beta-actin and alpha-enolase), have not, to our knowledge, earlier been implicated in schizophrenia pathology. Overall, these results with a combined approach of genomics and proteomics add to the validity of subchronic N-methyl-D-aspartate (NMDA)-receptor antagonist treatment as an animal model of schizophrenia.

Proteome analysis of cerebrospinal fluid proteins in Alzheimer patients.

By comparing the CSF proteome between Alzheimer disease (AD) patients and controls it may be possible to identify proteins that play a role in the disease process and thus to study the pathogenesis of AD. We used mini-gel technology in a two-dimensional electrophoresis procedure, sensitive SYPRO Ruby staining and mass spectrometry for clinical screening of disease-influenced CSF proteins in 15 AD patients and 12 controls. The levels of six proteins and their isoforms, including proapolipoprotein, apolipoprotein E, beta-2 microglobulin, retinol-binding protein, transthyretin, and ubiquitin, were significantly altered in CSF of AD patients. The most prominently altered proteins were the apolipoproteins, especially proapolipoprotein.