1.
Nilsson, C. L., et al.
(författare)
Use of ENCODE Resources to Characterize Novel Proteoforms and Missing Proteins in the Human Proteome
2015
Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 14:2, s. 603-608
Tidskriftsartikel (refereegranskat) abstract
We describe the utility of integrated strategies that employ both translation of ENCODE data and major proteomic technology pillars to improve the identification of the "missing proteins", novel proteoforms, and PTMs. On one hand, databases in combination with bioinformatic tools are efficiently utilized to establish microarray-based transcript analysis and supply rapid protein identifications in clinical samples. On the other hand, sequence libraries are the foundation of targeted protein identification and quantification using mass spectrometric and immunoaffinity techniques. The results from combining proteoENCODEdb searches with experimental mass spectral data indicate that some alternative splicing forms detected at the transcript level are in fact translated to proteins. Our results provide a step toward the directives of the C-HPP initiative and related biomedical research.
2.
Nilsson, C. L., et al.
(författare)
Chromosome 19 Annotations with Disease Speciation: A First Report from the Global Research Consortium
2013
Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 12:1, s. 134-149
Tidskriftsartikel (refereegranskat) abstract
A first research development progress report of the Chromosome 19 Consortium with members from Sweden, Norway, Spain, United States, China and India, a part of the Chromosome-centric Human Proteome Project (C-HPP) global initiative, is presented (http://www.c-hpp.org). From the chromosome 19 peptide-targeted library constituting 6159 peptides, a pilot study was conducted using a subset with 125 isotope-labeled peptides. We applied an annotation strategy with triple quadrupole, ESI-Qtrap, and MALDI mass spectrometry platforms, comparing the quality of data within and in between these instrumental set-ups. LC–MS conditions were outlined by multiplex assay developments, followed by MRM assay developments. SRM was applied to biobank samples, quantifying kallikrein 3 (prostate specific antigen) in plasma from prostate cancer patients. The antibody production has been initiated for more than 1200 genes from the entire chromosome 19, and the progress developments are presented. We developed a dedicated transcript microarray to serve as the mRNA identifier by screening cancer cell lines. NAPPA protein arrays were built to align with the transcript data with the Chromosome 19 NAPPA chip, dedicated to 90 proteins, as the first development delivery. We have introduced an IT-infrastructure utilizing a LIMS system that serves as the key interface for the research teams to share and explore data generated within the project. The cross-site data repository will form the basis for sample processing, including biological samples as well as patient samples from national Biobanks.
3.
Nilsson, Anna, et al.
(författare)
Fine Mapping the Spatial Distribution and Concentration of Unlabeled Drugs within Tissue Micro-Compartments Using Imaging Mass Spectrometry
2010
Ingår i: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 5:7, s. e11411-
Tidskriftsartikel (refereegranskat) abstract
Readouts that define the physiological distributions of drugs in tissues are an unmet challenge and at best imprecise, but are needed in order to understand both the pharmacokinetic and pharmacodynamic properties associated with efficacy. Here we demonstrate that it is feasible to follow the in vivo transport of unlabeled drugs within specific organ and tissue compartments on a platform that applies MALDI imaging mass spectrometry to tissue sections characterized with high definition histology. We have tracked and quantified the distribution of an inhaled reference compound, tiotropium, within the lungs of dosed rats, using systematic point by point MS and MS/MS sampling at 200 mu m intervals. By comparing drug ion distribution patterns in adjacent tissue sections, we observed that within 15 min following exposure, tiotropium parent MS ions (mass-to-charge; m/z 392.1) and fragmented daughter MS/MS ions (m/z 170.1 and 152.1) were dispersed in a concentration gradient (80 fmol-5 pmol) away from the central airways into the lung parenchyma and pleura. These drug levels agreed well with amounts detected in lung compartments by chemical extraction. Moreover, the simultaneous global definition of molecular ion signatures localized within 2-D tissue space provides accurate assignment of ion identities within histological landmarks, providing context to dynamic biological processes occurring at sites of drug presence. Our results highlight an important emerging technology allowing specific high resolution identification of unlabeled drugs at sites of in vivo uptake and retention.
4.
Sundström, Ingela, et al.
(författare)
In vivo investigation of brain and systemic ketobemidone metabolism
2010
Ingår i: The Analyst. - : Royal Society of Chemistry (RSC). - 0003-2654 .- 1364-5528. ; 135:2, s. 405-413
Tidskriftsartikel (refereegranskat) abstract
Ketobemidone metabolites have previously been identified in urine and plasma; here we show, for the first time, that norketobemidone and ketobemidone N-oxide are present in in vivo microdialysate from rat brain (striatum) after reverse microdialysis, suggesting striatal metabolism of ketobemidone. Ketobemidone metabolites were also identified in in vivo microdialysate samples from brain and blood, as well as in urine from rats, after subcutaneous administration of ketobemidone. Three Phase I metabolites (norketobemidone, ketobemidone N-oxide and hydroxymethoxyketobemidone) and three Phase II metabolites (glucuronic acid conjugates of ketobemidone, norketobemidone and hydroxymethoxyketobemidone) were identified in the microdialysates after subcutaneous administration. Coupled capillary liquid chromatography tandem mass spectrometry (LC-MS/MS) and SPE (boronate)-MS/MS were utilized for the analysis of the biological samples. The Phase I metabolites were identified by comparing the retention times and tandem mass spectra of the microdialysates with synthetic standards. The Phase II metabolites were identified by determination of exact masses and by comparing the tandem mass spectra of the microdialysates with those of synthetic standards for the aglycones. Hydroxyketobemidone, a catechol-type Phase I metabolite, was selectively isolated by solid-phase boronate-complexation but identified in urine alone. This work demonstrated that the in vivo microdialysis technique in combination with LC-MS/MS can be used to study the local metabolism of a drug in the brain.
5.
Zhang, Xiaoqun, et al.
(författare)
Repeated l-DOPA treatment increases c-fos and BDNF mRNAs in the subthalamic nucleus in the 6-OHDA rat model of Parkinson's disease
2006
Ingår i: Brain Research. - : Elsevier BV. - 0006-8993 .- 1872-6240. ; 27, s. 96-96
Tidskriftsartikel (refereegranskat) abstract
The subthalamic nucleus and the striatum are input regions of the basal ganglia. This study used the unilateral 6-OHDA rat model of Parkinson's disease to examine effects of l-DOPA on the expression of c-fos and BDNF mRNAs in these nuclei. Dopamine depletion per se did not affect c-fos or BDNF. Both a single and repeated injections of l-DOPA induced c-fos, but not BDNF, in the dopamine-depleted striatum. However, repeated l-DOPA treatment increased c-fos and BDNF in the dopamine-depleted subthalamic nucleus. These molecular adaptations may reflect changes in neuronal plasticity that underlie some therapeutic actions and/or side effects of l-DOPA in Parkinson's disease.
6.
Lodén, Henrik, et al.
(författare)
An introduction to MS imaging in drug discovery and development
2015
Ingår i: Bioanalysis. - : Future Science Ltd. - 1757-6180 .- 1757-6199. ; 7:20, s. 2621-2627
Tidskriftsartikel (refereegranskat) abstract
A vital process in drug discovery and development is to assess the absorption, distribution, metabolism, excretion and toxicology of potentially therapeutic compounds in the body. The potential utility of MS imaging has been demonstrated in many studies focusing on molecules including peptides, proteins and lipids. However, MS imaging also permits the direct analysis of drugs and drug metabolites in tissue samples without requiring the use of target-specific labels or reagents. Here, a brief technical description of the technique is presented along with examples of its usefulness at different stages of the drug discovery and development process including absorption, distribution, metabolism, excretion and toxicology, and blood-brain barrier drug penetration investigations.
7.
Nezhyva, Mariya, et al.
(författare)
Spatial multiomic insights into acute cocaine exposure
Annan publikation (övrigt vetenskapligt/konstnärligt) abstract
Recent studies provide compelling evidence that cocaine-induced neurotoxicity begins within hours of a single acute cocaine exposure. Despite this, a comprehensive understanding of the molecular alterations occurring in vivo within the reward system following such an exposure has been lacking. In this study, we developed an analytical workflow that combines mass spectrometry imaging with microscale proteomics of brain regions. Here, we present a multiomic perspective on the molecular consequences of acute cocaine exposure on the principal areas of the reward system and the hippocampus. Our findings include distinct region-specific alterations in the tricarboxylic acid (TCA) cycle and lipid synthesis within the reward circuitry highlighting a significant energy depletion in mice 24 hours post-cocaine injections. Additionally, we linked widespread reductions in key neurotransmitters (GABA, glutamate, aspartate) across the reward system and calcium level modifications to changes in synaptic plasticity and mitochondria dysfunction. Mitochondrial dysfunction and energy metabolism disruption were evident through imbalances in the mitochondrial ATP production and electron transport chain components, increased susceptibility to oxidative stress, disturbances in mitochondrial transport proteins, and fluctuations in creatine and taurine levels. Among the brain regions within the reward circuitry, the prefrontal cortex (PFC) exhibited the most pronounced effects. This study not only provides a holistic overview of the intricate interplay between proteins and metabolites within the reward circuitry regions during the onset of cocaine-induced neurotoxicity but also offers novel insights into the underlying molecular mechanisms.
8.
Végvári, Ákos, et al.
(författare)
Essential tactics of tissue preparation and matrix nano-spotting for successful compound imaging mass spectrometry
2010
Ingår i: Journal of Proteomics. - : Elsevier BV. - 1876-7737 .- 1874-3919. ; 73:6, s. 1270-1278
Tidskriftsartikel (refereegranskat) abstract
The ultimate goal of MALDI-Imaging Mass Spectrometry (MALDI-IMS) is to achieve spatial localization of analytes in tissue sections down to individual tissue compartments or even at the level of a few cells. With compound tissue imaging, it is possible to track the transportation of an unlabelled, inhaled reference compound within lung tissue, through the application of MALDI-IMS. The procedure for isolation and preparation of lung tissues is found to be crucial in order to preserve the anatomy and structure of the pulmonary compartments. To avoid delocalization of analytes within lung tissue compartments we have applied an in-house designed nano-spotter, based on a microdispenser mounted on an XY table, of which movement and spotting functionality were fully computer controlled. We demonstrate the usefulness of this platform in lung tissue sections isolated from rodent in vivo model, applied to compound tissue imaging as exemplified with the determination of the spatial distribution of (1 alpha,2 beta,4 beta,7 beta)-7-[(hydroxidi-2-thienylacetyl)oxy]-9,9-dimethyl-3-oxa-9-azoniatric yclo[3.3.1.0(2,4)]nonane, also known as tiotropium. We provide details on tissue preparation protocols and sample spotting technology for successful identification of drug in mouse lung tissue by using MALDI-Orbitrap instrumentation.
9.
Akhtar, Malik N., et al.
(författare)
Identification of best indicators of peptide-spectrum match using a permutation resampling approach
2014
Ingår i: Journal of Bioinformatics and Computational Biology. - 0219-7200 .- 1757-6334. ; 12:5, s. 1440001-
Tidskriftsartikel (refereegranskat) abstract
Various indicators of observed-theoretical spectrum matches were compared and the resulting statistical significance was characterized using permutation resampling. Novel decoy databases built by resampling the terminal positions of peptide sequences were evaluated to identify the conditions for accurate computation of peptide match significance levels. The methodology was tested on real and manually curated tandem mass spectra from peptides across a wide range of sizes. Spectra match indicators from complementary database search programs were pro filed and optimal indicators were identified. The combination of the optimal indicator and permuted decoy databases improved the calculation of the peptide match significance compared to the approaches currently implemented in the database search programs that rely on distributional assumptions. Permutation tests using p-values obtained from software-dependent matching scores and E-values outperformed permutation tests using all other indicators. The higher overlap in matches between the database search programs when using end permutation compared to existing approaches con firmed the superiority of the end permutation method to identify peptides. The combination of effective match indicators and the end permutation method is recommended for accurate detection of peptides.
10.
Alm, Henrik, et al.
(författare)
Exposure to brominated flame retardant PBDE-99 affects cytoskeletal protein expression in the neonatal mouse cerebral cortex
2008
Ingår i: Neurotoxicology. - : Elsevier BV. - 0161-813X .- 1872-9711. ; 29:4, s. 628-637
Tidskriftsartikel (refereegranskat) abstract
Polybrominated diphenyl ethers (PBDEs) are environmental contaminants found in human and animal tissues worldwide. Neonatal exposure to the flame retardant 2,2', 4,4',5-pentabromodiphenyl ether (PBDE-99) disrupts normal brain development in mice, and results in disturbed spontaneous behavior in the adult. The mechanisms underlying the late effects of early exposure are not clear. To gain insight into the initial neurodevelopmental damage inflicted by PBDE-99, we investigated the short-term effects of PBDE-99 on protein expression in the developing cerebral cortex of neonatal mice, and the cytotoxic and apoptotic effects of PBDE-99 in primary cultures of fetal rat cortical cells. We used two-dimensional difference gel electrophoresis (2D-DIGE) to analyze protein samples isolated from the cortex of NMRI mice 24h after exposure to a single oral dose of 12 mg/kg PBDE-99 on post-natal day 10. Protein resolution was enhanced by sample pre-fractionation. In the cell model, we determined cell viability using the trypan blue exclusion assay, and apoptosis using immunocytochemical detection of cleaved caspase-3. We determined the identity of 111 differentially expressed proteins, 32 (29%) of which are known to be cytoskeleton-related. Similar to previous findings in the striatum, we found elevated levels of the neuron growth-associated protein Gap43 in the cortex. In cultured cortical cells, a high concentration of PBDE-99 (30 microM) induced cell death without any apparent increase in caspase-3 activity. These results indicate that the permanent neurological damage induced by PBDE-99 during the brain growth spurt involve detrimental effects on cytoskeletal regulation and neuronal maturation in the developing cerebral cortex.
