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  • 1.
    Alm, Henrik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Toxicology.
    Kultima, Kim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Toxicology.
    Scholz, Birger
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Toxicology.
    Nilsson, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Andrén, Per E
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Fex-Svenningsen, Åsa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Dencker, Lennart
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Toxicology.
    Stigson, Michael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Toxicology.
    Exposure to brominated flame retardant PBDE-99 affects cytoskeletal protein expression in the neonatal mouse cerebral cortex2008In: Neurotoxicology, ISSN 0161-813X, E-ISSN 1872-9711, Vol. 29, no 4, p. 628-637Article in journal (Refereed)
    Abstract [en]

    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.

  • 2.
    Dahlin, Andreas P.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Bergström, Sara K.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Andrén, Per E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Markides, Karin E.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Poly(dimethylsiloxane)-Based Microchip for Two-Dimensional Solid-Phase Extraction-Capillary Electrophoresis with an Integrated Electrospray Emitter Tip2005In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 77, no 16, p. 5356-5363Article in journal (Refereed)
    Abstract [en]

    A microchip in poly(dimethylsiloxane) (PDMS) for in-line solid-phase extraction-capillary electrophoresis-electrospray ionization-time-of-flight mass spectrometry (SPE-CE-ESI-TOF-MS) has been developed and evaluated. The chip was fabricated in a novel one-step procedure where mixed PDMS was cast over steel wires in a mold. The removed wires defined 50-um cylindrical channels. Fused-silica capillaries were inserted into the structure in a tight fit connection. The inner walls of the inserted fused-silica capillaries and the PDMS microchip channels were modified with a positively charged polymer, PolyE-323. The chip was fabricated in a two-level cross design. The channel at the lower level was packed with 5-um hyper-cross-linked polystyrene beads acting as a SPE medium used for desalting. The upper level channel acted as a CE channel and ended in an integrated emitter tip coated with conducting graphite powder to facilitate the electrical contact for sheathless ESI. An overpressure continuously provided fresh CE electrolyte independently of the flows in the different levels. Further studies were carried out in order to investigate the electrophoretic and flow rate properties of the chip. Finally, six-peptide mixtures, in different concentrations, dissolved in physiological salt solution was injected, desalted, separated, and sprayed into the mass spectrometer for analysis with a limit of detection in femtomole levels.

  • 3. Frank, Ari M.
    et al.
    Savitski, Mikhail M.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Nielsen, Michael L.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Zubarev, Roman A.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Pevzner, Pavel A.
    De novo peptide sequencing and identification with precision mass spectrometry2007In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 6, no 1, p. 114-123Article in journal (Refereed)
    Abstract [en]

    The recent proliferation of novel mass spectrometers such as Fourier transform, QTOF, and OrbiTrap marks a transition into the era of precision mass spectrometry, providing a 2 orders of magnitude boost to the mass resolution, as compared to low-precision ion-trap detectors. We investigate peptide de novo sequencing by precision mass spectrometry and explore some of the differences when compared to analysis of low-precision data. We demonstrate how the dramatically improved performance of de novo sequencing with precision mass spectrometry paves the way for novel approaches to peptide identification that are based on direct sequence lookups, rather than comparisons of spectra to a database. With the direct sequence lookup, it is not only possible to search a database very efficiently, but also to use the database in novel ways, such as searching for products of alternative splicing or products of fusion proteins in cancer. Our de novo sequencing software is available for download at http://peptide.ucsd.edu/.

  • 4.
    Fälth, Maria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Savitski, Mikhail M.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Nielsen, Michael L.
    Kjeldsen, Frank
    Andrén, Per E
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Zubarev, Roman A.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    SwedCAD, a database of annotated high-mass accuracy MS/MS spectra of tryptic peptides2007In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 6, no 10, p. 4063-4067Article in journal (Refereed)
    Abstract [en]

    A database of high-mass accuracy tryptic peptides has been created. The database contains 15897 unique, annotated MS/MS spectra. It is possible to search for peptides according to their mass, number of missed cleavages, and sequence motifs. All of the data contained in the database is downloadable, and each spectrum can be visualized. An example is presented of how the database can be used for studying peptide fragmentation. Fragmentation of different types of missed cleaved peptides has been studied, and the results can be used to improve identification of these types of peptides.

  • 5.
    Fälth, Maria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Sköld, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Norrman, Mathias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Svensson, Marcus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Fenyö, David
    Andrén, Per E
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    SwePep – A database designed for endogenous peptides and mass spectrometry2006In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 5, no 6, p. 998-1005Article in journal (Refereed)
    Abstract [en]

    A new database, SwePep, specifically designed for endogenous peptides, has been constructed to significantly speed up the identification process from complex tissue samples utilizing mass spectrometry. In the identification process the experimental peptide masses are compared with the peptide masses stored in the database both with and without possible post-translational modifications. This intermediate identification step is fast and singles out peptides that are potential endogenous peptides and can later be confirmed with tandem mass spectrometry data. Successful applications of this methodology are presented. The SwePep database is a relational database developed using MySql and Java. The database contains 4180 annotated endogenous peptides from different tissues originating from 394 different species as well as 50 novel peptides from brain tissue identified in our laboratory. Information about the peptides, including mass, isoelectric point, sequence, and precursor protein, is also stored in the database. This new approach holds great potential for removing the bottleneck that occurs during the identification process in the field of peptidomics. The SwePep database is available to the public.

  • 6.
    Fälth, Maria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Sköld, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Norrman, Mathias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Svensson, Marcus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Fenyö, David
    Andrén, Per E
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    SwePep, a database designed for endogenous peptides and mass spectrometry2006In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 5, no 6, p. 998-1005Article in journal (Refereed)
    Abstract [en]

    A new database, SwePep, specifically designed for endogenous peptides, has been constructed to significantly speed up the identification process from complex tissue samples utilizing mass spectrometry. In the identification process the experimental peptide masses are compared with the peptide masses stored in the database both with and without possible post-translational modifications. This intermediate identification step is fast and singles out peptides that are potential endogenous peptides and can later be confirmed with tandem mass spectrometry data. Successful applications of this methodology are presented. The SwePep database is a relational database developed using MySql and Java. The database contains 4180 annotated endogenous peptides from different tissues originating from 394 different species as well as 50 novel peptides from brain tissue identified in our laboratory. Information about the peptides, including mass, isoelectric point, sequence, and precursor protein, is also stored in the database. This new approach holds great potential for removing the bottleneck that occurs during the identification process in the field of peptidomics. The SwePep database is available to the public.

  • 7.
    Fälth, Maria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Sköld, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Svensson, Marcus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Nilsson, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Fenyö, David
    Andrén, Per E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Neuropeptidomics strategies for specific and sensitive identification of endogenous peptides2007In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 6, no 7, p. 1188-1197Article in journal (Refereed)
    Abstract [en]

    A new approach using targeted sequence collections has been developed for identifying endogenous peptides. This approach enables a fast, specific, and sensitive identification of endogenous peptides. Three different sequence collections were constituted in this study to mimic the peptidomic samples: SwePep precursors, SwePep peptides, and SwePep predicted. The searches for neuropeptides performed against these three sequence collections were compared with searches performed against the entire mouse proteome, which is commonly used to identify neuropeptides. These four sequence collections were searched with both Mascot and X! Tandem. Evaluation of the sequence collections was achieved using a set of manually identified and previously verified peptides. By using the three new sequence collections, which more accurately mimic the sample, 3 times as many peptides were significantly identified, with a false-positive rate below 1%, in comparison with the mouse proteome. The new sequence collections were also used to identify previously uncharacterized peptides from brain tissue; 27 previously uncharacterized peptides and potentially bioactive neuropeptides were identified. These novel peptides are cleaved from the peptide precursors at sites that are characteristic for prohormone convertases, and some of them have post-translational modifications that are characteristic for neuropeptides. The targeted protein sequence collections for different species are publicly available for download from SwePep.

  • 8. Kaplan, Anders
    et al.
    Söderström, Malin
    Fenyö, David
    Nilsson, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Fälth, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Sköld, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Svensson, Marcus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Pettersen, Harald
    Lindqvist, Staffan
    Svenningsson, Per
    Andrén, Per E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Björkesten, Lennart
    An Automated Method for Scanning LC−MS Data Sets for Significant Peptides and Proteins, Including Quantitative Profiling and Interactive Confirmation: An Automated Method for Scanning LC−MS Data Sets for Significant Peptides and Proteins, Including Quantitative Profiling and Interactive Confirmation2007In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 6, no 7, p. 2888-2895Article in journal (Refereed)
    Abstract [en]

    Differential quantification of proteins and peptides by LC-MS is a promising method to acquire knowledge about biological processes, and for finding drug targets and biomarkers. However, differential protein analysis using LC-MS has been held back by the lack of suitable software tools. Large amounts of experimental data are easily generated in protein and peptide profiling experiments, but data analysis is time-consuming and labor-intensive. Here, we present a fully automated method for scanning LC-MS/MS data for biologically significant peptides and proteins, including support for interactive confirmation and further profiling. By studying peptide mixtures of known composition, we demonstrate that peptides present in different amounts in different groups of samples can be automatically screened for using statistical tests. A linear response can be obtained over almost 3 orders of magnitude, facilitating further profiling of peptides and proteins of interest. Furthermore, we apply the method to study the changes of endogenous peptide levels in mouse brain striatum after administration of reserpine, a classical model drug for inducing Parkinson disease symptoms.

  • 9.
    Kjeldsen, Frank
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Savitski, Mikhail
    Adams, Christopher
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Zubarev, Roman
    Determination of the location of positive charges in gas-phase polypeptide polycations by tandem mass spectrometry2006In: International Journal of Mass Spectrometry, ISSN 1387-3806, E-ISSN 1873-2798, Vol. 252, no 3, p. 204-212Article in journal (Refereed)
    Abstract [en]

    Location of protonated sites in electrospray-ionized gas-phase peptides and proteins was performed with tandem mass spectrometry using ion activation by both electron capture dissociation (ECD) and collisional activation dissociation (CAD). Charge-carrying sites were assigned based on the increment in the charge state of fragment ions compared to that of the previous fragment in the same series. The property of ECD to neutralize preferentially the least basic site was confirmed by the analysis of three thousand ECD mass spectra of doubly charged tryptic peptides. Multiply charged cations of bradykinin, neurotensin and melittin were studied in detail. For n+ precursors, ECD revealed the positions of (n − 1) most basic sites, while CAD could in principle locate all n charges. However, ECD introduced minimal proton mobilization and produced more conclusive data than CAD, for which N- and C-terminal data often disagreed. Consistent with the dominance of one charge conformer and its preservation in ECD, the average charge states of complementary fragments of n+ ions almost always added up to (n − 1)+, while the similar figure in CAD often deviated from n+, indicating extensive charge isomerization under collisional excitation. For bradykinin and neurotensin, the charge assignments were largely in agreement with the intrinsic gas-phase basicity of the respective amino acid residues. For melittin ions in higher charge states, ECD revealed the charging at both intrinsically basic as well as at less basic residues, which was attributed to charge sharing with other groups due to the presence of secondary and higher order structures in this larger polypeptide.

  • 10.
    Kjeldsen, Frank
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Savitski, Mikhail M.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Nielsen, Michael L.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Shi, L.
    Zubarev, Roman A.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    On studying protein phosphorylation patterns using bottom-up LC-MS/MS: the case of human alpha-casein2007In: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 132, no 8, p. 768-776Article in journal (Refereed)
    Abstract [en]

    Most proteomics studies involving mapping post-translational modifications, such as the phosphorylation of serine and threonine, are performed today using the 'bottom-up' approach. This approach involves enzymatic cleavage of proteins, most often by trypsin, with subsequent nano-LC-MS/MS. The occupancy rates of phosphosites in proteins may differ by orders of magnitude, and thus the occupancy rate must be reported for each occupied phosphosite. To highlight potential pitfalls in quantifying the occupancy rates, αs1- casein from human milk was selected as a model molecule representing moderately phosphorylated proteins. For this purpose, human milk from one Caucasian woman in the eighth month of lactation was used. The phosphorylation level of caseins is believed to have major implications for the formation of micelles that are involved in delivering valuable calcium phosphate and other minerals to the new-born. Human αs1-casein has been reported to be much less phosphorylated than ruminant caseins, which may indicate a different function of caseins in humans. Revealing the phosphorylation pattern in human casein can thus shed light on its function. The current study found that the sequence region between the residues Ser70 and Ser76 in human αs1-casein is in fact phosphorylated, contrary to previous knowledge. The site of the most abundant phosphorylation is Ser75, in agreement with the known action of the mammary gland casein kinase. There is evidence for the second phosphorylation in that region, possibly at Ser73. Earlier reported positions of phosphorylations at Ser18 and Ser26 are also confirmed, but not the dominance of Ser18 phosphorylation. The occupancy rates at Ser18, Ser26 and Ser75 are estimated to be (7 ± 2), (20 ± 6) and (27 ± 9)%, respectively. Owing to differences in the ionization efficiency between phosphorylated and unphosphorylated peptides a 30% error margin is added to the occupancy rates. The highlighted pitfalls of the bottom-up strategy include the sensitivity of enzymes to proximal acidic and phosphorylated residues and the presence of multiple isoforms, including unexpected ones, of the tryptic peptides. The utility of the earlier introduced PhosTS_hunter and ModifiComb approaches for evading the latter pitfall is demonstrated.

  • 11.
    Klintenberg, Rebecka
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Andrén, Per E
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Altered extracellular striatal in vivo biotransformation of the opioid neuropeptide dynorphin A(1-17) in the unilateral 6-OHDA rat model of Parkinson's disease2005In: Journal of Mass Spectrometry, ISSN 1076-5174, E-ISSN 1096-9888, Vol. 40, no 2, p. 261-270Article in journal (Refereed)
    Abstract [en]

    The in vivo biotransformation of dynorphin A(1-17) (Dyn A) was studied in the striatum of hemiparkinsonian rats by using microdialysis in combination with nanoflow reversed-phase liquid chromatography/electrospray time-of-flight mass spectrometry. The microdialysis probes were implanted into both hemispheres of unilaterally 6-hydroxydopamine (6-OHDA) lesioned rats. Dyn A (10 pmol microl(-1)) was infused through the probes at 0.4 microl min(-1) for 2 h. Samples were collected every 30 min and analyzed by mass spectrometry. The results showed for the first time that there was a difference in the Dyn A biotransformation when comparing the two corresponding sides of the brain. Dyn A metabolites 1-8, 1-16, 5-17, 10-17, 7-10 and 8-10 were detected in the dopamine-depleted striatum but not in the untreated striatum. Dyn A biotransformed fragments found in both hemispheres were N-terminal fragments 1-4, 1-5, 1-6, 1-11, 1-12 and 1-13, C-terminal fragments 2-17, 3-17, 4-17, 7-17 and 8-17 and internal fragments 2-5, 2-10, 2-11, 2-12, and 8-15. The relative levels of these fragments were lower in the dopamine-depleted striatum. The results imply that the extracellular in vivo processing of the dynorphin system is being disturbed in the 6-OHDA-lesion animal model of Parkinson's disease.

  • 12. Lerner, Mikael
    et al.
    Corcoran, Martin
    Cepeda, Diana
    Nielsen, Michael L.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Zubarev, Roman
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Pontén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Uhlén, Mathias
    Hober, Sophia
    Grandér, Dan
    Sangfelt, Olle
    The RBCC Gene RFP2 (Leu5) Encodes a Novel Transmembrane E3 Ubiquitin Ligase Involved in ERAD2007In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 18, no 5, p. 1670-1682Article in journal (Refereed)
    Abstract [en]

    RFP2, a gene frequently lost in various malignancies, encodes a protein with RING finger, B-box, and coiled-coil domains that belongs to the RBCC/TRIM family of proteins. Here we demonstrate that Rfp2 is an unstable protein with auto-polyubiquitination activity in vivo and in vitro, implying that Rfp2 acts as a RING E3 ubiquitin ligase. Consequently, Rfp2 ubiquitin ligase activity is dependent on an intact RING domain, as RING deficient mutants fail to drive polyubiquitination in vitro and are stabilized in vivo. Immunopurification and tandem mass spectrometry enabled the identification of several putative Rfp2 interacting proteins localized to the endoplasmic reticulum (ER), including valosin-containing protein (VCP), a protein indispensable for ER-associated degradation (ERAD). Importantly, we also show that Rfp2 regulates the degradation of the known ER proteolytic substrate CD3-δ, but not the N-end rule substrate Ub-R-YFP (yellow fluorescent protein), establishing Rfp2 as a novel E3 ligase involved in ERAD. Finally, we show that Rfp2 contains a C-terminal transmembrane domain indispensable for its localization to the ER and that Rfp2 colocalizes with several ER-resident proteins as analyzed by high-resolution immunostaining. In summary, these data are all consistent with a function for Rfp2 as an ERAD E3 ubiquitin ligase.

  • 13.
    Madeira, Alexandra
    et al.
    Karolinska Institutet .
    Vikeved, Elisabet
    Karolinska Institutet.
    Nilsson, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Sjögren, Benita
    Karolinska Institutet.
    Andrén, Per E
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Svenningsson, Per
    Karolinska Institutet.
    Identification of protein-protein interactions by surface plasmon resonance followed by mass spectrometry2011In: Current protocols in protein science, ISSN 1934-3663, Vol. Suppl. 65, p. 19.21.1-19.21.9Article in journal (Refereed)
    Abstract [en]

    Elucidation of the function and meaning of the protein networks can be useful in the understanding of many pathological processes and the identification of new therapeutic targets. This unit describes an approach to discover protein-protein interactions by coupling surface plasmon resonance to mass spectrometry. Briefly, a protein is covalently bound to a sensor chip, which is then exposed to brain extracts injected over the surface via a microfluidic system. This allows the monitoring in real-time of the interactions between the immobilized ligand and the extracts. Interacting proteins from the extracts are then recovered, trypsinized, and identified using mass spectrometry. The data obtained are searched against a sequence database using the Mascot software. To exclude nonspecific interactors, control experiments using blank sensor chips, and/or randomized peptides, are performed. The protocol presented here does not require specific labeling or modification of proteins and can be performed in <4 days.

  • 14.
    Medin, Sofia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Quantitative analysis of acetylcholine in mouse brain tissue sections by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Imaging2014Independent thesis Advanced level (degree of Master (One Year)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Intro             This study revolves around the process of aging, with respect to acetylcholine production, which declines with increasing age and can cause dementia-related diseases. The acetylcholinesterase inhibitor tacrine was previously used in the treatment of Alzheimer’s disease and was used to study its effect with respect to increased acetylcholine production in young (12 weeks) and old (14 months) mice.

    Method         Duplicates of frozen brain tissue were cut into sagittal and coronal sections with a thickness of 14 µm, and mounted on conductive indium tin oxide (ITO) glass slides. A total of four animals were used, one young tacrine treated, one old tacrine treated, one young control and one old control. Imaging and quantitation acetylcholine was performed using Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Imaging (MALDI MSI).

    Results, Discussion and Conclusion     This study presents a proof-of-principle of the quantitation of acetylcholine using MALDI MS Imaging. The results showed a 3-fold increase in acetylcholine in young mice, and a 2-fold increase in the old mice when comparing tacrine treated and control. The areas of higher concentrations correspond to the presynaptic neurons at the termination of the cholinergic pathways, which include the cortex, hippocampus, thalamus, hypothalamus and hindbrain. One exception is the cerebellum, which exhibited low concentrations in all tissues. The distribution of acetylcholine in the control mice was more evenly distributed. A tacrine dependent increase of acetylcholine in the basolateral amygdala was detected in both young and old mice, an area involved in fear-related learning. Another nucleus called the interpeduncular nucleus (IPN) exhibited a 3-4 fold increase compared to the rest of the midbrain. IPN was unaffected by the tacrin treatment and observed in all animals. This is explained by the dense cholinergic innervation from the medial habenula, which is an important link between forebrain and midbrain structures. 

  • 15.
    Nielsen, Michael L.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Savitski, Mikhail M.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Zubarev, Roman A.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Extent of modifications in human proteome samples and their effect on dynamic range of analysis in shotgun proteomics2006In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 5, no 12, p. 2384-2391Article in journal (Refereed)
    Abstract [en]

    The complexity of the uman proteome, already enormous at the organism level, increases further in the course of the proteome analysis due to in vitro sample evolution. Most of in vitro alterations can also occur in vivo as post-translational modifications. These two types of modifications can only be distinguished a posteriori but not in the process of analysis, thus rendering necessary the analysis of every molecule in the sample. With the new software tool ModifiComb applied to MS/MS data, the extent of modifications was measured in tryptic mixtures representing the full proteome of human cells. The estimated level of 8-12 modified peptides per each unmodified tryptic peptide present at ≥1 % level is approaching one modification per amino acid on average. This is a higher modification rate than was previously thought, posing an additional challenge to analytical techniques. The solution to the problem is seen in improving sample preparation routines, introducing dynamic range-adjusted thresholds for database searches, using more specific MS/MS analysis using high mass accuracy and complementary fragmentation techniques, and revealing peptide families with identification of additional proteins only by unfamiliar peptides. Extensive protein separation prior to analysis reduces the requirements on speed and dynamic range of a tandem mass spectrometer and can be a viable alternative to the shotgun approach.

  • 16.
    Patriksson, Alexandra
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Adams, Christopher M.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Kjeldsen, Frank
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Zubarev, Roman
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    van der Spoel, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    A direct comparison of protein structure in the gas and solution phase - the Trp-cage2007In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 111, no 46, p. 13147-13150Article in journal (Refereed)
    Abstract [en]

    Molecular dynamics simulations of zwitterions of the Trp-cage protein in the gas phase show that the most stable ion in vacuo has preserved the charge locations acquired in solution. A direct comparison of the gas and solution-phase structures reveals that, despite the similarity in charge location, there is significant difference in the structures, with a substantial increase in hydrogen bonds and exposure of hydrophobic parts in the gas phase. The structure of the salt bridge in the gas phase is also much more stable than in the (experimental) solution structure.

  • 17. Petruzziello, Filomena
    et al.
    Fouillen, Laetitia
    Wadensten, Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Kretz, Robert
    Andrén, Per E
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Rainer, Gregor
    Zhang, Xiaozhe
    Extensive Characterization of Tupaia belangeri Neuropeptidome Using an Integrated Mass Spectrometric Approach2012In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 11, no 2, p. 886-896Article in journal (Refereed)
    Abstract [en]

    Neuropeptidomics is used to characterize endogenous peptides in the brain of tree shrews (Tupaia belangeri). Tree shrews are small animals similar to rodents in size but close relatives of primates, and are excellent models for brain research. Currently, tree shrews have no complete proteome information available on which direct database search can be allowed for neuropeptide identification. To increase the capability in the identification of neuropeptides in tree shrews, we developed an integrated mass spectrometry (MS)-based approach that combines methods including data-dependent, directed, and targeted liquid chromatography (LC)-Fourier transform (FT)-tandem MS (MS/MS) analysis, database construction, de novo sequencing, precursor protein search, and homology analysis. Using this integrated approach, we identified 107 endogenous peptides that have sequences identical or similar to those from other mammalian species. High accuracy MS and tandem MS information, with BLAST analysis and chromatographic characteristics were used to confirm the sequences of all the identified peptides. Interestingly, further sequence homology analysis demonstrated that tree shrew peptides have a significantly higher degree of homology to equivalent sequences in humans than those in mice or rats, consistent with the close phylogenetic relationship between tree shrews and primates. Our results provide the first extensive characterization of the peptidome in tree shrews, which now permits characterization of their function in nervous and endocrine system. As the approach developed fully used the conservative properties of neuropeptides in evolution and the advantage of high accuracy MS, it can be portable for identification of neuropeptides in other species for which the fully sequenced genomes or proteomes are not available.

  • 18.
    Pierson, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Svenningsson, Per
    Caprioli, Richard M.
    Andrén, Per E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Increased Levels of Ubiquitin in the 6-OHDA-Lesioned Striatum of Rats2005In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 4, no 2, p. 223-226Article in journal (Refereed)
    Abstract [en]

    Multiple genetic deficits have linked impaired ubiquitin-conjugation pathways to various forms of familiar Parkinson's disease. We therefore examined the possible role of 6-hydroxydopamine, a dopaminergic neurotoxin used in Parkinson's disease experimental models, in causing protein degradation and its association with the ubiquitin proteasome system. Using unilaterally 6-hydroxydopamine-denervated rats and mass spectrometry profiling directly on brain tissue sections, we here report for the first time an increased level of unconjugated ubiquitin specifically in the dorsal striatum of the dopamine depleted hemisphere. No similar changes were found in the intact hemisphere or in the ventral striatum of the dopamine depleted hemisphere. The lesioning of the dopamine innervation to the striatum was confirmed by a strongly reduced dopamine transporter binding in the striatum, indicating an abundant loss of dopamine neurons. These results suggest that denervation of dopamine neurons per se is implicated in the regulation of ubiquitin pathways, at least in a classical animal model of Parkinson's disease. This study adds additional information regarding the involvement of the ubiquitin-proteasome system in Parkinson's disease.

  • 19. Rand, Kasper D.
    et al.
    Adams, Christopher M.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Zubarev, Roman A.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Jorgensen, Thomas J. D.
    Electron capture dissociation proceeds with a low degree of intramolecular migration of peptide amide hydrogens2008In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 130, no 4, p. 1341-1349Article in journal (Refereed)
    Abstract [en]

    Hydrogen (H-1/H-2) exchange combined with mass spectrometry (HX-MS) has become a recognized method for the analysis of protein structural dynamics. Presently, the incorporated deuterons are typically localized by enzymatic cleavage of the labeled proteins and single residue resolution is normally only obtained for a few residues. Determination of site-specific deuterium levels by gas-phase fragmentation in tandem mass spectrometers would greatly increase the applicability of the HX-MS method. The biggest obstacle in achieving this goal is the intramolecular hydrogen migration (i.e., hydrogen scrambling) that occurs during vibrational excitation of gas-phase ions. Unlike traditional collisional ion activation, electron capture dissociation (ECD) is not associated with substantial vibrational excitation. We investigated the extent of intramolecular backbone amide hydrogen (H-1/H-2) migration upon ECD using peptides with a unique selective deuterium incorporation. Our results show that only limited amide hydrogen migration occurs upon ECD, provided that vibrational excitation prior to the electron capture event is minimized. Peptide ions that are excessively vibrationally excited in the electrospray ion source by, e.g., high declustering potentials or during precursor ion selection (via side band excitation) in the external linear quadrupole ion trap undergo nearly complete hydrogen (H-1/H-2) scrambling. Similarly, collision-induced dissociation (CID) in the external linear quadrupole ion trap results in complete or extensive hydrogen (H-1/H-2) scrambling. This precludes the use of CID as a method to obtain site-specific information from proteins that are labeled in solution-phase H-1/H-2 exchange experiments. In contrast, the deuteration levels of the c- and z-fragment ions generated from ECD closely mimic the known solution deuteration pattern of the selectively labeled peptides. This excellent correlation between the results obtained from gas phase and solution suggests that ECD holds great promise as a general method to obtain single residue resolution in proteins from solution H-1/H-2 exchange experiments.

  • 20. Samgina, T. Yu.
    et al.
    Arternenko, K. A.
    Gorshkov, V. A.
    Lebedev, A.
    Nielsen, Michael L.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Savitski, Mikhail M.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Zubarev, Roman
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Electrospray ionization tandem mass spectrometry sequencing of novel skin peptides from Ranid frogs containing disulfide bridges2007In: European journal of mass spectrometry, ISSN 1469-0667, E-ISSN 1751-6838, Vol. 13, no 2, p. 155-163Article in journal (Refereed)
    Abstract [en]

    Tandem mass spectrometry sequencing, as well as Edman sequencing of peptides belonging to the Rana genus, represents a difficult task due to the presence of a disulfide bridge at the C-terminus and their rather high molecular masses (over 2000Da). The present study throws light upon the sequence of three rather long peptides (more than 20 amino acid residues each) isolated from the skin secretion of Russian frogs, Rana ridibunda and Rana arvalis. This novel aspect involves the fact that the sequences (including two sequences established de novo) were determined exclusively by means of mass spectrometry. A combination of electron capture dissociation (ECD) and collision-induced dissociaiton (CID) data accompanied by exact mass measurements (LTQ Fourier transform ion cyclotron resonance mass spectrometer) facilitated reaching the goal. To overcome the difficulty dealing with disulphide bridges ("Rana box"), reduction of the S-S bond with dithiotreitol followed by derivatization of Cys residues with iodoacetamide was used. The sequence was determined using combined spectral data on y and b series of fragment ions. A multiple mass spectrometry (MS') experiment was also used to elucidate the sequence inside the "Rana box" after cysteine derivatization. Exact mass measurements were used to differentiate between Lys and Gln residues, while characteristic losses of 29 and 43 Da (d and w fragment ions) in CID and ECD experiments allowed us to distinguish between Ile and Leu isomeric acids.

  • 21.
    Savitski, Mikhail
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Kjeldsen, Frank
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Nielsen, Michael L.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Zubarev, Roman A.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Hydrogen Rearrangement to and from Radical z Fragments in Electron Capture Dissociation of Peptides2007In: Journal of the American Society for Mass Spectrometry, ISSN 1044-0305, E-ISSN 1879-1123, Vol. 18, no 1, p. 113-120Article in journal (Refereed)
    Abstract [en]

    Hydrogen rearrangement is an important process in radical chemistry. A high degree of H· rearrangement to and from z· ionic fragments (combined occurrence frequency 47% compared with that of z·) is confirmed in analysis of 15,000 tandem mass spectra of tryptic peptides obtained with electron capture dissociation (ECD), including previously unreported double H· losses. Consistent with the radical character of H· abstraction, the residue determining the formation rate of z′ = z· + H· species is found to be the N-terminal residue in z· species. The size of the complementary cm′ fragment turned out to be another important factor, with z′ species dominating over z· ions for m ≤ 6. The H· atom was found to be abstracted from the side chains as well as from α-carbon groups of residues composing the c′ species, with Gln and His in the c′ fragment promoting H· donation and Asp and Ala opposing it. Ab initio calculations of formation energies of ·A radicals (A is an amino acid) confirmed that the main driving force for H· abstraction by z· is the process exothermicity. No valid correlation was found between the N{single bond}Cα bond strength and the frequency of this bond cleavage, indicating that other factors than thermochemistry are responsible for directing the site of ECD cleavage. Understanding hydrogen attachment to and loss from ECD fragments should facilitate automatic interpretation ECD mass spectra in protein identification and characterization, including de novo sequencing.

  • 22.
    Savitski, Mikhail M.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Kjeldsen, Frank
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Nielsen, Michael L.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Garbuzynskiy, Sergiy O.
    Galzitskaya, Oxana V.
    Surin, Alexey K.
    Zubarev, Roman A.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Backbone carbonyl group basicities are related to gas-phase fragmentation of peptides and protein folding2007In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 46, no 9, p. 1481-1484Article in journal (Refereed)
    Abstract [en]

    A strong correlation is found between the propensity of individual amino acids to induce peptide-bond cleavage in the gas phase (PAA-XX) and their structure-forming propensity (PS, red) and H-bond-accepting propensity (PH, blue). Thus, the same fundamental parameter, carbonyl group basicity, governs the formation of secondary protein structures in solution and directs fragmentation in the gas phase. (Graph Presented).

  • 23.
    Savitski, Mikhail M.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Kjeldsen, Frank
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Nielsen, Michael L.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Zubarev, Roman A.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Relative specificities of water and ammonia losses from backbone fragments in collision-activated dissociation2007In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 6, no 7, p. 2669-2673Article in journal (Refereed)
    Abstract [en]

    Analysis of a database containing over 20 000 high-resolution collision-activation mass spectra of tryptic peptide dications was employed to study the relative specificity of neutral losses from backbone fragments. The high resolution of the FTMS instrument allowed for the first time the first isotope of the water loss and the monoisotope of the ammonia loss to be distinguished. Contrary to a popular belief, water losses from y' ions are not specific enough to rely upon for detecting the presence of amino acids with oxygen in the side chains. At the same time, ammonia loss from b ions is sufficiently specific (> 95%) to detect the presence of amino acids Gln, Asn, His, Lys, and Arg. This feature will be useful for de novo algorithms for high-resolution MS data. Clear trends were observed when the effect of amino acids proximate to the cleavage site on the rate of loss formation was studied. These trends turned out to be different for losses from b and y ions.

  • 24.
    Savitski, Mikhail M.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Nielsen, Michael L.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Zubarev, Roman A.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Side-chain losses in electron capture dissociation to improve peptide identification2007In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 79, no 6, p. 2296-2302Article in journal (Refereed)
    Abstract [en]

    Analysis of a database of some 20 000 conventional electron-capture dissociation (ECD) mass spectra of doubly charged ions belonging to tryptic peptides revealed widespread appearance of w ions and related u ions that are due to partial side chain losses from radical z. ions. Half of all z. ions that begin with Leu or Ile produce w ions in conventional one-scan ECD mass spectra, which differentiates these isomeric residues with >97% reliability. Other residues exhibiting equally frequent side chain losses are Gln, Glu, Asp, and Met (cysteine was not included in this work). Unexpectedly, Asp lost not a radical group like other amino acids but a molecule CO2, thus giving rise to a radical w. ion with the possibility of a radical cascade. Losses from amino acids as distant as seven residues away from the cleavage site were detected. The mechanism of such losses seems to be related to radical migration from the original site at the αCn atom in a Zn. ion to other αC and βC atoms. The side chain losses confirm sequence assignment, improve the database matching score, and can be useful in de novo sequencing.

  • 25.
    Sundström, Ingela
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Andrén, Per E
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Westerlund, Douglas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Method development for identification of ketobemidone metabolites in microdialysate samples by coupled-column capillary liquid chromatography-tandem mass spectrometry2008In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1189, no 1-2, p. 503-513Article in journal (Refereed)
    Abstract [en]

    Methodologies for identification of ketobemidone metabolites in microdialysate samples utilizing coupled-column capillary liquid chromatography-electrospray quadrupole time-of-flight tandem mass spectrometry are presented. Two different methods were developed to efficiently analyze the metabolites norketobemidone, ketobemidone N-oxide and hydroxyketobemidone, respectively. Both methods include on-line desalting and trapping of the analytes on micro-solid-phase extraction columns with different retention mechanisms. Norketobemidone and ketobemidone N-oxide were trapped on a C18 column and then eluted by back-flush followed by isocratic separation on a fluorinated reversed-phase type silica gel column (Fluofix). Retention equations are proposed for the chromatographic observations made on the Fluofix column. Hydroxyketobemidone was trapped on a phenylboronic acid column by complex formation at basic pH and then eluted at acidic pH directly into to the mass spectrometer. Oxidation of hydroxyketobemidone to its corresponding quinone was also observed. The methods were successfully used to analyze synthetic ketobemidone metabolites in dilute low-volume microdialysis samples

  • 26.
    Svensson, Marcus
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Sköld, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Nilsson, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Fälth, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Nydahl, Katarina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Svenningsson, Per
    Andrén, Per E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Neuropeptidomics: MS applied to the discovery of novel peptides from the brain2007In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 79, no 1, p. 14-21Article in journal (Refereed)
    Abstract [en]

    Peptidomics involves the comprehensive analysis of the peptide content of a certain cell, organ, body fluid, or organism. Per E. Andrén and colleagues at Uppsala University and the Karolinska Institutet (both in Sweden) describe neuropeptidomics approaches in brain lysates and tissue sections to study peptide expression in disease models and to identify potentially biologically active neuropeptides.

  • 27. Tarasova, I A
    et al.
    Gorshkov, A V
    Evreinov, V V
    Adams, K
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Zubarev, Roman A
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Gorshkov, M V
    Applicability of the critical chromatography concept to proteomics problems: Experimental study of the dependence of peptide retention time on the sequence of amino acids in the chain2008In: Polymer science, ISSN 0965-545X, Vol. 50, no 3, p. 309-321Article in journal (Refereed)
    Abstract [en]

    Experimental data on the separation of synthetic and natural peptides are presented as treated in terms of the separation model proposed by the authors, which allows for the chain connectivity of amino acid residues and the cooperative character of their interaction with the surface. It was shown that the model accurately predicts the separation of peptides with identical amino acid contents and different sequences of units in the chain. The differences in the sequence may be permutation of amino acid residues and the presence of terminal groups, amino acid isomers, or mirror sequences in the chain. The separation model was used to predict the retention times of peptides prepared via the enzymatic hydrolysis of E. coli proteins and bovine serum albumin with trypsin. It was shown that in general the model accurately explains the array of experimental data on the separation of such peptides, thus being the first successful attempt to relate the chain sequence to the retention volume.

  • 28.
    Zhang, Xiaoqun
    et al.
    Karolinska Institutet, Institutionen för fysiologi och farmakologi.
    Andren, Per E
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Chergui, Karima
    Karolinska Institutet, Institutionen för fysiologi och farmakologi.
    Svenningsson, Per
    Karolinska Institutet, Institutionen för fysiologi och farmakologi.
    Neurokinin B/NK3 receptors exert feedback inhibition on L-DOPA actions in the 6-OHDA lesion rat model of Parkinson's disease2008In: Neuropharmacology, ISSN 0028-3908, E-ISSN 1873-7064, Vol. 54, no 7, p. 1143-1152Article in journal (Refereed)
    Abstract [en]

    Neurokinin B (NKB) and substance P (SP) act via NK(3) and NK(1) receptors. Using the unilateral 6-hydroxydopamine (6-OHDA) lesion rat model of Parkinson's disease (PD), it was found that chronic, but not acute, administration of L-DOPA increases striatal NKB expression in the dopamine-depleted hemisphere. In contrast, both acute and chronic administrations of L-DOPA restore reduced levels of SP mRNA. Co-treatment with the NK(3) receptor antagonist, SB222200, and L-DOPA increased contralateral rotations compared to L-DOPA alone in L-DOPA primed rats. The NK(3)R agonist, senktide, increased the phosphorylation of tyrosine hydroxylase (TH) at Ser(19)-TH, a CaMKII site, and of Thr(286)-CaMKII in striatal slices. Senktide had no effect on P-Ser(31)-TH, a MAPK site, but reduced P-Ser(217/221)-MEK. Amperometry demonstrated that senktide increased evoked dopamine release. SB222200 blocked the effects of senktide. In striatal slices prepared from 6-OHDA-lesioned rats repeatedly treated with L-DOPA, senktide no longer increased P-Thr(286)-CaMKII, suggesting a role of NK(3)R on dopamine terminals under normal conditions. SB222200 increased P-Ser(217/221)-MEK only in dopamine-depleted slices, indicating an increased NK(3)R tone under Parkinsonism conditions. Altogether, these data demonstrate a differential regulation of NKB and SP by L-DOPA in an animal model of PD and indicate a unique role of NKB in long-term effects of L-DOPA. Behavioural, biochemical and amperometric data indicate that NKB/NK(3)R signalling stimulates dopamine transmission at the presynaptic site, but inhibits it at the postsynaptic site. The inhibitory influence of NKB/NK(3)R on dopamine transmission dominates in an animal model of PD and provides a feedback inhibition on actions mediated via L-DOPA.

  • 29.
    Zhang, Xiaoqun
    et al.
    Karolinska Institutet, Institutionen för fysiologi och farmakologi.
    Andren, Per E
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Greengard, Paul
    The Rockefeller University, Laboratory of Molecular and Cellular Neuroscience.
    Svenningsson, Per
    Karolinska Institutet, Institutionen för fysiologi och farmakologi.
    Evidence for a role of the 5-HT1B receptor and its adaptor protein, p11, in L-DOPA treatment of an animal model of Parkinsonism2008In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 6, p. 2163-2168Article in journal (Refereed)
    Abstract [en]

    Parkinson's disease (PD) is characterized by a progressive degeneration of substantia nigra dopaminergic neurons projecting to the striatum. Restoration of dopamine transmission by L-DOPA relieves symptoms of PD but causes prominent side effects. There is a strong serotonin innervation of the striatum by serotonergic neurons that remains relatively preserved in PD. The study of this innervation has been largely neglected. Here, we demonstrate that chronic L-DOPA administration to 6-OHDA-lesioned rodents increases, via D1 receptors, the levels of the 5-HT1B receptor and its adaptor protein, p11, in dopamine-denervated striatonigral neurons. Using unilaterally 6-OHDA-lesioned p11 WT and KO mice, it was found that administration of a selective 5-HT1B receptor agonist, CP94253, inhibited L-DOPA-induced rotational behavior and abnormal involuntary movements in a p11-dependent manner. These data reveal an L-DOPA-induced negative-feedback mechanism, whereby the serotonin system may influence the symptomatology of Parkinsonism.

  • 30. Zhang, Xiaoqun
    et al.
    Andrén, Per E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Svenningsson, Per
    Changes on 5-HT2 receptor mRNAs in striatum and subthalamic nucleus in Parkinson's disease model2007In: Physiology and Behavior, ISSN 0031-9384, E-ISSN 1873-507X, Vol. 92, no 1-2, p. 29-33Article in journal (Refereed)
    Abstract [en]

    Changes on 5-HT2 receptor mRNAs in striatum and subthalamic nucleus in Parkinson's disease model. PHYSIOL. BEHAV. 00(0), 000-000, 2006. Abnormal interactions between the serotonin and dopamine systems may underlie the high prevalence of non-motor complications in Parkinson's disease (PD). Here, we demonstrate that the genes encoding serotonin 5-HT2A and 5-HT2C receptors are differently regulated by dopamine in the 6-hydroxydopamine (6-OHDA) rat model of PD. Nigrostriatal cell loss causes an up-regulation of 5-HT2AR mRNA, but a down-regulation of 5-HT2CR mRNA, in striatum. Repeated injections with L-DOPA/benserazide reverse the effect of 6-OHDA lesioning on 5-HT2AR, but not on 5-HT2CR, gene expression. Neither 6-OHDA-lesioning nor L-DOPA/benserazide treatment had any effect on 5-HT2AR mRNA in cortex or on 5-HT2CR mRNA in nucleus subthalamicus. These data suggest that the regulation of 5-HT2AR in striatum, in the 6-OHDA rat model of PD, is mainly dependent upon alterations in dopamine levels. 5-HT2CR, on the other hand, are regulated by nigrostriatal cell loss and by the accompanied reduction of factor(s), other than dopamine, that are normally co-expressed with dopamine. The apparent imbalance between 5-HT2AR and 5-HT2CR levels in this PD model indicates a potential role for these receptors in the pathophysiology of neuropsychiatric symptoms, such as depression and L-DOPA-induced hallucinations, which are co-morbid with PD. The fact that 5-HT2CR are differentially regulated as compared to 5-HT2AR to alterations in the dopamine tone predicts that pharmacological manipulations at 5-HT2CR, but not at 5-HT2AR, will result in similar effects in PD patients whether they are treated or not with dopamine replacement.

  • 31.
    Zhang, Xiaoqun
    et al.
    Karolinska Institutet, Institutionen för fysiologi och farmakologi .
    Andrén, Per E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Svenningsson, Per
    Karolinska Institutet, Institutionen för fysiologi och farmakologi.
    Repeated l-DOPA treatment increases c-fos and BDNF mRNAs in the subthalamic nucleus in the 6-OHDA rat model of Parkinson's disease2006In: Brain Research, ISSN 0006-8993, E-ISSN 1872-6240, Vol. 1095, p. 207-210Article in journal (Refereed)
    Abstract [en]

    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.

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