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  • 1. Akhtar, Malik N.
    et al.
    Southey, Bruce R.
    Andrén, Per E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Sweedler, Jonathan V.
    Rodriguez-Zas, Sandra L.
    Evaluation of Database Search Programs for Accurate Detection of Neuropeptides in Tandem Mass Spectrometry Experiments2012In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 11, no 12, p. 6044-6055Article in journal (Refereed)
    Abstract [en]

    Neuropeptide identification in mass spectrometry experiments using database search programs developed for proteins is challenging. Unlike proteins, the detection of the complete sequence using a single spectrum is required to identify neuropeptides or prohormone peptides. This study compared the performance of three open-source programs used to identify proteins, OMSSA, X!Tandem and Crux, to identify prohormone peptides. From a target database of 7850 prohormone peptides, 23550 query spectra were simulated across different scenarios. Crux was the only program that correctly matched all peptides regardless of p-value and at p-value < 1 X 10(-2), 33%, 64%, and >75%, of the 5, 6, and >= 7 amino acid-peptides were detected. Crux also had the best performance in the identification of peptides from chimera spectra and in a variety of missing ion scenarios. OMSSA, X!Tandem and Crux correctly detected 98.9% (99.9%), 93.9% (97.4%) and 88.7% (98.3%) of the peptides at E- or p-value < 1 X 10(-6) (< 1 X 10(-2)), respectively. OMSSA and X! Tandem outperformed the other programs in significance level and computational speed, respectively. A consensus approach is not recommended because some prohormone peptides were only identified by one program.

  • 2.
    Alm, Henrik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Scholz, Birger
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Kultima, Kim
    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.
    Andrén, Per E
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Savitski, Mikhail M
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Bergman, Åke
    Stigson, Michael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Fex-Svenningsen, Åsa
    Institute of Medical Biology, Anatomy and Neurobiology, University of Southern Denmark, Denmark.
    Dencker, Lennart
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    In Vitro Neurotoxicity of PBDE-99: Immediate and Concentration-Dependent Effects on Protein Expression in Cerebral Cortex Cells2010In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 9, no 3, p. 1226-1235Article in journal (Refereed)
    Abstract [en]

    Polybrominated diphenyl ethers (PBDEs) are commonly used flame retardants in various consumer products. Pre- and postnatal exposure to congeners of PBDEs disrupts normal brain development in rodents. Two-dimensional difference gel electrophoresis (2D-DIGE) was used to analyze concentration-dependent differences in protein expression in cultured cortical cells isolated from rat fetuses (GD 21) after 24 h exposure to PBDE-99 (3, 10, or 30 muM). Changes on a post-translational level were studied using a 1 h exposure to 30 muM PBDE-99. The effects of 24 h exposure to 3 and 30 muM PBDE-99 on mRNA levels were measured using oligonucleotide microarrays. A total of 62, 46, and 443 proteins were differentially expressed compared to controls after 24 h of exposure to 3, 10, and 30 muM PDBE-99, respectively. Of these, 48, 43, and 238 proteins were successfully identified, respectively. We propose that the biological effects of low-concentration PBDE-99 exposure are fundamentally different than effects of high-concentration exposure. Low-dose PBDE-99 exposure induced marked effects on cytoskeletal proteins, which was not correlated to cytotoxicity or major morphological effects, suggesting that other more regulatory aspects of cytoskeletal functions may be affected. Interestingly, 0.3 and 3 muM, but not 10 or 30 muM increased the expression of phosphorylated (active) Gap43, perhaps reflecting effects on neurite extension processes.

  • 3. Amelina, Hanna
    et al.
    Apraiz, Itxaso
    Sun, Wei
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Cristobal, Susana
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Proteomics-based method for the assessment of marine pollution using liquid chromatography coupled with two-dimensional electrophoresis2007In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 6, no 6, p. 2094-2104Article in journal (Refereed)
    Abstract [en]

    Using a proteomic approach, we have developed a new method for the assessment of marine pollution that generates highly reproducible protein expression patterns and it is simple and scalable. The protocol is based on applying liquid chromatography ( LC) coupled with two-dimensional electrophoresis (2DE) to analyze changes in the protein expression pattern after exposure to marine pollution. The digestive gland of the sentinel "blue mussel" ( Mytilus edulis) was batch-processed through a simple cell fractionation followed by ion-exchange chromatography and 2-DE. The selection of ligands, elution method, and small volume design was carefully considered to define a protocol that could be mainly robotized. A pilot study with samples collected from different Gothenburg harbor areas indicated that the clean area could be distinguished from the polluted ones based on a protein expression pattern ( PES) composed of 13 proteins. Principal component analysis ( PCA) and hierarchical clustering confirmed that the PES was sufficient to discriminate polluted and unpolluted areas and to provide a spatial gradient from the polluted source. Several proteins from the PES were identified by electrospray ionization tandem mass spectrometry ( ESI-MS/MS), and they are involved in,beta-oxidation, amino acid metabolism, detoxification, protein degradation, organelle biogenesis, and protein folding. In the near future, this methodology could show potential advantages to assess marine pollution and could become a stable platform to elucidate ecotoxicological questions.

  • 4. Bakshi, Mayur V
    et al.
    Barjaktarovic, Zarko
    Azimzadeh, Omid
    Kempf, Stefan J.
    Merl, Juliane
    Hauck, Stefanie M.
    Buratovic, Sonja
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Environmental toxicology.
    Eriksson, Per
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Environmental toxicology.
    Atkinson, Michael J.
    Tapio, Soile
    Total body exposure to low-dose ionizing radiation induces long term alterations to the liver proteome of neonatally exposed mice2015In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 14, no 1, p. 366-373Article in journal (Refereed)
    Abstract [en]

    Tens of thousands of people are being exposed daily toenvironmental low-dose gamma radiation. Epidemiological data indicate thatsuch low radiation doses may negatively affect liver function and result in thedevelopment of liver disease. However, the biological mechanisms behindthese adverse effects are unknown. The aim of this study was to investigateradiation-induced damage in the liver after low radiation doses. Neonatal maleNMRI mice were exposed to total body irradiation on postnatal day 10 usingacute single doses ranging from 0.02 to 1.0 Gy. Early (1 day) and late (7months) changes in the liver proteome were tracked using isotope-codedprotein label technology and quantitative mass spectrometry. Our dataindicate that low and moderate radiation doses induce an immediateinhibition of the glycolysis pathway and pyruvate dehydrogenase availability inthe liver. Furthermore, they lead to significant long-term alterations in lipidmetabolism and increased liver inflammation accompanying inactivation of thetranscription factor peroxisome proliferator-activated receptor alpha. This study contributes to the understanding of the potentialrisk of liver damage in populations environmentally exposed to ionizing radiation.

  • 5.
    Bergström Lind, Sara
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Molin, Magnus
    Savitski, Mikhail M.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Emilsson, Lina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Åström, Jonas
    Uppsala BIO.
    Hedberg, Ludwig
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Adams, Chris
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Nielsen, Michael
    Engström, Åke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Elfineh, Lioudmila
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Andersson, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Zubarev, Roman
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Immunoaffinity Enrichments Followed by Mass Spectrometric Detection for Studying Global Protein Tyrosine Phosphorylation2008In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 7, no 7, p. 2897-2910Article in journal (Refereed)
    Abstract [en]

    Phosphorylation of protein tyrosine residues regulates important cell functions and is, when dysregulated, often crucially involved in oncogenesis. It is therefore important to develop and evaluate methods for identifying and studying tyrosine phosphorylated (P-Tyr) proteins. P-Tyr proteins are present at very low concentrations within cells, requiring highly selective enrichment methods to be detected. In this study, we applied immunoaffinity as enrichment step for P-Tyr proteins. Five selected anti-phosphotyrosine antibodies (monoclonal antibodies 4G10, PY100, PYKD1, 13F9 and one polyclonal antiserum) were evaluated with respect to their capability to enrich P-Tyr proteins from cell extracts of the K562 leukemia cell line. The enrichment resulted in the detection of a group of proteins that potentially were tyrosine-phosphorylated (putative P-Tyr proteins). High accuracy identification of actual P-Tyr sites were performed using a highly selective and sensitive liquid chromatography Fourier transform mass spectrometer (LC-FTMS) setup with complementary collision activated dissociation (CAD) and electron capture dissociation (ECD) fragmentations. 4G10 and PY100 antibodies recognized the greatest number of putative P-Tyr proteins in initial screening experiments and were therefore further evaluated and compared in immunoaffinity enrichment of both P-Tyr proteins and peptides. Using the 4G10 antibody for enrichment of proteins, we identified 459 putative P-Tyr proteins by MS. Out of these proteins, 12 were directly verified as P-Tyr proteins by MS analysis of the actual site. Using the PY100 antibody for enrichment of peptides, we detected 67 P-Tyr peptides (sites) and 89 putative P-Tyr proteins. Generally, enrichment at the peptide level made it difficult to reliably determine the identity of the proteins. In contrast, protein identification following immunoaffinity enrichment at the protein level gave greater sequence coverage and thus a higher confidence in the protein identification. By combining all available information, 40 proteins were identified as true P-Tyr proteins from the K562 cell line. In conclusion, this study showed that a combination of immunoaffinity enrichment using multiple antibodies of both intact and digested proteins in parallel experiments is required for best possible coverage of all possible P-Tyr proteins in a sample.

  • 6.
    Carapito, Christine
    et al.
    Univ Strasbourg, Lab Spectrometrie Masse BioOrgan LSMBO, IPHC, CNRS,UMR7178, 25 Rue Becquerel, F-67087 Strasbourg, France..
    Duek, Paula
    SIB, CALIPHO Grp, CMU, Rue Michel Servet 1, CH-1211 Geneva 4, Switzerland..
    Macron, Charlotte
    Univ Strasbourg, Lab Spectrometrie Masse BioOrgan LSMBO, IPHC, CNRS,UMR7178, 25 Rue Becquerel, F-67087 Strasbourg, France..
    Seffals, Marine
    Univ Rennes 1, UMS BioSit, Core Facil H2P2, F-35040 Rennes, France..
    Rondel, Karine
    Irset, INSERM, U1085, Protim, Campus Beaulieu, F-35042 Rennes, France..
    Delalande, Francois
    Univ Strasbourg, Lab Spectrometrie Masse BioOrgan LSMBO, IPHC, CNRS,UMR7178, 25 Rue Becquerel, F-67087 Strasbourg, France..
    Lindskog, Cecilia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Freour, Thomas
    CHU Nantes, Serv Med Reprod, 38 Blvd Jean Monnet, F-44093 Nantes, France.;INSERM, UMR1064, F-44093 Nantes, France..
    Vandenbrouck, Yves
    CEA, DRF, BIG, Lab Biol Grande Echelle, 17 Rue Martyrs, F-38054 Grenoble, France.;INSERM, U1038, F-38054 Grenoble, France.;Grenoble Alpes Univ, F-38054 Grenoble, France..
    Lane, Lydie
    SIB, CALIPHO Grp, CMU, Rue Michel Servet 1, CH-1211 Geneva 4, Switzerland.;Univ Geneva, Fac Med, Dept Human Prot Sci, 1 Rue Michel Servet, CH-1211 Geneva 4, Switzerland..
    Pineau, Charles
    Irset, INSERM, U1085, Protim, Campus Beaulieu, F-35042 Rennes, France..
    Validating Missing Proteins in Human Sperm Cells by Targeted Mass-Spectrometry- and Antibody-based Methods2017In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 16, no 12, p. 4340-4351Article in journal (Refereed)
    Abstract [en]

    The present study is a contribution to the "neXt50 challenge", a coordinated effort across C-HPP teams to identify the SO most tractable missing proteins (MPs) on each chromosome. We report the targeted search of 38 theoretically detectable MPs from chromosomes 2 and 14 in Triton X-100 soluble and insoluble sperm fractions from a total of 15 healthy donors. A targeted mass spectrometry-based strategy consisting of the development of LC-PRM assays (with heavy labeled synthetic peptides) targeting 92 proteotypic peptides of the 38 selected MPs was used. Out of the 38 selected MPs, 12 were identified with two or more peptides and 3 with one peptide after extensive SDS-PAGE fractionation of the two samples and with overall low-intensity signals. The PRM data are available via ProteomeXchange in PASSEL (PASS01013). Further validation by immunohistochemistry on human testes sections and cytochemistry on sperm smears was performed for eight MPs with antibodies available from the Human Protein Atlas. Deep analysis of human sperm still allows the validation of MPs and therefore contributes to the C-HPP worldwide effort. We anticipate that our results will be of interest to the reproductive biology community because an in-depth analysis of these MPs may identify potential new candidates in the context of human idiopathic infertilities.

  • 7. Chorell, Ellin
    et al.
    Moritz, Thomas
    Branth, Stefan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Antti, Henrik
    Svensson, Michael B.
    Predictive Metabolomics Evaluation of Nutrition-Modulated Metabolic Stress Responses in Human Blood Serum During the Early Recovery Phase of Strenuous Physical Exercise2009In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 8, no 6, p. 2966-2977Article in journal (Refereed)
    Abstract [en]

    We have investigated whether postexercise ingestion of carbohydrates in combination with proteins generates a different systemic metabolic response, as compared to the sole ingestion of carbohydrate or water, in the early recovery phase following exercise. In addition, metabolic patterns related to fitness level were studied together with individual responses to nutritional modulation. Twenty-four male subjects were exposed to 90 min of ergometer-cycling. Each participant was subject to four identical test-sessions, including ingestion of one of four beverages (water, low-carbohydrate beverage, high-carbohydrate beverage, and low-carbohydrate-protein beverage (LCHO-P)) immediately after cycling. Blood was collected at six time points, one pre- and five postexercise. Extracted blood serum was subject to metabolomic characterization by gas chromatography/time-of-flight mass spectrometry (GC-TOF MS). Data was processed using hierarchical multivariate curve resolution (HMCR), and multivariate statistical analysis was carried out using orthogonal partial least-squares (OPLS). Predictive metabolomics, including predictive HMCR and OPLS classification, was applied to ensure efficient sample processing and validation of detected metabolic patterns. Separation of subjects in relation to ingested beverage was detected and interpreted. Pseudouridine was suggested as a novel marker for pro-anabolic effect following LCHO-P ingestion, which was supported by the detected decrease of the catabolic marker 3-methylhistidine. Separation of subjects according to fitness level was achieved, and nutritional modulation by LCHO-P was shown to improve the metabolic status of less fit subjects in the recovery phase. In addition, the potential of the methodology for detection of early signs of insulin resistance was also demonstrated.

  • 8.
    Chowdhury, Azazul
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Satagopam, Venkata P.
    Manukyan, Levon
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Artemenko, Konstantin A.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Fung, Yi Man Eva
    Schneider, Reinhard
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Bergsten, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Signaling in Insulin-Secreting MIN6 Pseudoislets and Monolayer Cells2013In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 12, no 12, p. 5954-5962Article in journal (Refereed)
    Abstract [en]

    Cell cell interactions are of fundamental importance for cellular function. In islets of Langerhans, which control blood glucose levels by secreting insulin in response to the blood . glucose concentration, the secretory response of intact islets is c higher than that of insulin-producing beta-cells not arranged in the islet architecture. The objective was to define mechanisms by which cellular performance is enhanced when cells are arranged in a) three-dimensional space. The task was addressed by making a c comprehensive analysis based on protein expression patterns " generated from insulin-secreting MIN6 cells grown as islet-like c clusters, so-called pseudoislets, and in monolayers. After culture, glucose-stimulated insulin secretion (GSIS) was measured from monolayers and pseudoislets. GSIS rose 6-fold in pseudoislets but only 3-fold in monolayers when the glucose concentration was increased from 2 to 20 mmol/L. Proteins from pseudoislets and monolayers were extracted and analyzed by liquid-chromatography mass spectrometry, and differentially expressed proteins were mapped onto KEGG pathways. Protein profiling identified 1576 proteins, which were common to pseudoislets and monolayers. When mapped onto KEGG pathways, 11 highly enriched pathways were identified. On the basis of differences in expression of proteins belonging to the pathways in pseudoislets and monolayers, predictions of differential pathway activation were performed. Mechanisms enhancing insulin secretory capacity of the beta-cell, when situated in the islet, include pathways regulating glucose metabolism, cell interaction, and translational regulation.

  • 9.
    Conrotto, Paolo
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Yakymovych, Ihor
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Yakymovych, Mariya
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Souchelnytskyi, Serhiy
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Interactome of transforming growth factor-beta type I receptor (T beta RI): Inhibition of TGF beta signaling by Epac12007In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 6, no 1, p. 287-297Article in journal (Refereed)
    Abstract [en]

    Transforming growth factor-beta (TGF beta) is a potent regulator of cell growth, differentiation, and apoptosis. Type I TGF beta receptor (T beta RI) is the key receptor for initiation of intracellular signaling by TGF beta. Here we report proteomics-based identification of proteins that form a complex with T beta RI. Using 2D-GE and MALDI TOF mass spectrometry, we identified 16 proteins that specifically interacted with a GST-fused T beta RI Thr204Asp construct with constitutively active serine/threonine kinase. We confirmed interactions of the receptor with cAMP regulated guanine nucleotide exchange factor 1 (Epac1), alpha-spectrin, PIASy, and alpha-catenin proteins using immunoblotting. Interaction of the receptor with Epac1 required intact kinase activity of T beta RI but was not affected by deletion of cAMP-binding domain of Epac1. TGF beta 1-induced C-terminal phosphorylation of Smad2 was inhibited in vivo and in vitro in the presence of Epac1. Epac1 inhibited also TGF beta 1/T beta RI-dependent transcriptional activation, as evaluated by luciferase reporter assays. We observed that expression of Epac1 counteracted TGF beta/T beta RI-dependent decrease of cell adhesion and TGF beta/T beta RI-induced stimulation of cell migration. Thus, we have reported novel T beta RI-interacting proteins and have shown that Epac1 inhibited TGF beta-dependent regulation of cell migration and adhesion.

  • 10.
    Ekegren, Titti
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Hanrieder, Jörg
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Aquilonius, Sten-Magnus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Neurology.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Focused proteomics in post-mortem human spinal cord2006In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 5, no 9, p. 2364-2371Article in journal (Refereed)
    Abstract [en]

    With a highly sensitive electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) system, proteins were identified in minimal amounts of spinal cord from patients with the neurodegenerative disease amyotrophic lateral sclerosis (ALS) and compared to proteins in spinal cord from control subjects. The results show 18 versus 16 significantly identified ( p < 0.05) proteins, respectively, all known to be found in the central nervous system. The most abundant protein in both groups was the glial fibrillary acidic protein, GFAP. Other proteins were, for example, hemoglobin alpha- and, chain, myelin basic protein, thioredoxin, R enolase, and cholin acetyltransferase. This study also includes the technique of laser microdissection in combination with pressure catapulting (LMPC) for the dissection of samples and specific neurons. Furthermore, complementary experiments with nanoLC-matrix assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS) confirmed the results of the ESI-FTICR MS screening and provided additional results of further identified proteins.

  • 11.
    Ekman, Martin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Ow, Saw Yen
    ChELSI Institute, Department of Chemical and Process Engineering, The University of Sheffield.
    Holmqvist, Marie
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Zhang, Xiaohui
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    van Wagenen, Jon
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Wright, Phillip C.
    ChELSI Institute, Department of Chemical and Process Engineering, The University of Sheffield.
    Stensjö, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Metabolic Adaptations in a H-2 Producing Heterocyst-Forming Cyanobacterium: Potentials and Implications for Biological Engineering2011In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 10, no 4, p. 1772-1784Article in journal (Refereed)
    Abstract [en]

    Nostoc punctiforme ATCC 29133 is a photoautotrophic cyanobacterium with the ability to fix atmospheric nitrogen and photoproduce hydrogen through the enzyme nitrogenase. The H-2 produced is reoxidized by an uptake hydrogenase. Inactivation of the uptake hydrogenase in N. punctiforme leads to increased H-2 release but unchanged rates of N-2 fixation, indicating redirected metabolism. System-wide understanding of the mechanisms of this metabolic redirection was obtained using complementary quantitative proteomic approaches, at both the filament and the heterocyst level. Of the total 1070 identified and quantified proteins, 239 were differentially expressed in the uptake hydrogenase mutant (NHM5) as compared to wild type. Our results indicate that the inactivation of uptake hydrogenase in N. punctiforme changes the overall metabolic equilibrium, affecting both oxygen reduction mechanisms in hetcrocysts as well as processes providing reducing equivalents for metabolic functions such as N-2 fixation. We identify specific metabolic processes used by NHM5 to maintain a high rate of N-2 fixation, and thereby potential targets for further improvement of nitrogenase based H-2 photogeneration. These targets include, but are not limited to, components of the oxygen scavenging capacity and cell envelope of heterocysts and proteins directly or indirectly involved in reduced carbon transport from vegetative cells to heterocysts.

  • 12.
    Eriksson, Cecilia
    et al.
    KTH, Proteomik.
    Agaton, Charlotta
    KTH, Skolan för bioteknologi (BIO).
    Kånge, Rikard
    Sundberg, Marten
    KTH, Skolan för bioteknologi (BIO).
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO).
    Ek, Bo
    Uhlen, Mathias
    KTH, Proteomik.
    Gustafsson, Magnus
    Hober, Sophia
    KTH, Proteomik.
    Microfluidic analysis of antibody specificity in a compact disk format2006In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 5, no 7, p. 1568-1574Article in journal (Refereed)
    Abstract [en]

    A new and flexible technology for high throughput analysis of antibody specificity and affinity is presented. The method is based on microfluidics and takes advantage of compact disks (CDs) in which the centrifugal force moves fluids through microstructures containing immobilized metal affinity chromatography columns. Analyses are performed as a sandwich assay, where antigen is captured to the column via a genetically attached His(6)-tag. The antibodies to be analyzed are applied onto the columns. Thereafter, fluorescently labeled secondary antibodies recognize the bound primary antibodies, and detection is carried out by laser-induced fluorescence. The CDs contain 104 microstructures enabling analysis of antibodies against more than 100 different proteins using a single CD. Importantly, through the three- dimensional visualization of the binding patterns in a column it is possible to separate high affinity from low affinity binding. The method presented here is shown to be very sensitive, flexible and reproducible.

  • 13. Fagerberg, Linn
    et al.
    Oksvold, Per
    Skogs, Marie
    Algenäs, Cajsa
    Lundberg, Emma
    Pontén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Sivertsson, Asa
    Odeberg, Jacob
    Klevebring, Daniel
    Kampf, Caroline
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Asplund, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Sjöstedt, Evelina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Al-Khalili Szigyarto, Cristina
    Edqvist, Per-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Olsson, IngMarie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Rydberg, Urban
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Hudson, Paul
    Ottosson Takanen, Jenny
    Berling, Holger
    Björling, Lisa
    Tegel, Hanna
    Rockberg, Johan
    Nilsson, Peter
    Navani, Sanjay
    Jirström, Karin
    Mulder, Jan
    Schwenk, Jochen M
    Zwahlen, Martin
    Hober, Sophia
    Forsberg, Mattias
    von Feilitzen, Kalle
    Uhlén, Mathias
    Contribution of Antibody-based Protein Profiling to the Human Chromosome-centric Proteome Project (C-HPP)2013In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 12, no 6, p. 2439-2448Article in journal (Refereed)
    Abstract [en]

    A gene-centric Human Proteome Project has been proposed to characterize the human protein-coding genes in a chromosome-centered manner to understand human biology and disease. Here, we report on the protein evidence for all genes predicted from the genome sequence based on manual annotation from literature (UniProt), antibody-based profiling in cells, tissues and organs and analysis of the transcript profiles using next generation sequencing in human cell lines of different origins. We estimate that there is good evidence for protein existence for 69% (n = 13985) of the human protein-coding genes, while 23% have only evidence on the RNA level and 7% still lack experimental evidence. Analysis of the expression patterns shows few tissue-specific proteins and approximately half of the genes expressed in all the analyzed cells. The status for each gene with regards to protein evidence is visualized in a chromosome-centric manner as part of a new version of the Human Protein Atlas ( www.proteinatlas.org ).

  • 14. Fagerberg, Linn
    et al.
    Strömberg, Sara
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    El-Obeid, Adila
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Gry, Marcus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Nilsson, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Uhlen, Mathias
    Pontén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Asplund, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Large-Scale Protein Profiling in Human Cell Lines Using Antibody-Based Proteomics2011In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 10, no 9, p. 4066-4075Article in journal (Refereed)
    Abstract [en]

    Human cancer cell lines grown in vitro are frequently used to decipher basic cell biological phenomena and to also specifically study different forms of cancer. Here we present the first large-scale study of protein expression patterns in cell lines using an antibody-based proteomics approach. We analyzed the expression pattern of 5436 proteins in 45 different cell lines using hierarchical clustering, principal component analysis, and two-group comparisons for the identification of differentially expressed proteins. Our results show that immunohistochemically determined protein profiles can categorize cell lines into groups that overall reflect the tumor tissue of origin and that hematological cell lines appear to retain their protein profiles to a higher degree than cell lines established from solid tumors. The two-group comparisons reveal well-characterized proteins as well as previously unstudied proteins that could be of potential interest for further investigations. Moreover, multiple myeloma cells and cells of myeloid origin were found to share a protein profile, relative to the protein profile of lymphoid leukemia and lymphoma cells, possibly reflecting their common dependency of bone marrow microenvironment. This work also provides an extensive list of antibodies, for which high-resolution images as well as validation data are available on the Human Protein Atlas (www.proteinatlas.org), that are of potential use in cell line studies.

  • 15. 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/.

  • 16.
    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.

  • 17.
    Fälth, Maria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Svensson, Marcus
    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.
    Sköld, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Fenyö, David
    Andrén, Per E
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Validation of endogenous peptide identifications using a database of tandem mass spectra2008In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 7, no 7, p. 3049-3053Article in journal (Refereed)
    Abstract [en]

    The SwePep database is designed for endogenous peptides and mass spectrometry. It contains information about the peptides such as mass, p/, precursor protein and potential post-translational modifications. Here, we have improved and extended the SwePep database with tandem mass spectra, by adding a locally curated version of the global proteome machine database (GPMDB). In peptidomic experiment practice, many peptide sequences contain multiple tandem mass spectra with different quality. The new tandem mass spectra database in SwePep enables validation of low quality spectra using high quality tandem mass spectra. The validation is performed by comparing the fragmentation patterns of the two spectra using algorithms for calculating the correlation coefficient between the spectra. The present study is the first step in developing a tandem spectrum database for endogenous peptides that can be used for spectrum-to-spectrum identifications instead of peptide identifications using traditional protein sequence database searches.

  • 18.
    Groebe, Karlfried
    et al.
    Pivot Biomed Sci GmbH, D-54296 Trier, Germany.
    Cen, Jing
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Schvartz, Domitille
    Univ Geneva, Ctr Med Univ, Human Prot Sci Dept, CH-1211 Geneva, Switzerland.
    Sargsyan, Ernest
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Chowdhury, Azazul Islam
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Roomp, Kirsten
    Univ Luxembourg, Luxembourg Ctr Syst Biomed, L-4365 Esch Sur Alzette, Luxembourg.
    Schneider, Reinhard
    Univ Luxembourg, Luxembourg Ctr Syst Biomed, L-4365 Esch Sur Alzette, Luxembourg.
    Alderborn, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Sanchez, Jean-Charles
    Univ Geneva, Ctr Med Univ, Human Prot Sci Dept, CH-1211 Geneva, Switzerland.
    Bergsten, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Palmitate-Induced Insulin Hypersecretion and Later Secretory Decline Associated with Changes in Protein Expression Patterns in Human Pancreatic Islets2018In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 17, no 11, p. 3824-3836Article in journal (Refereed)
    Abstract [en]

    In obese children with high circulating concentrations of free fatty acid palmitate, we have observed that insulin levels at fasting and in response to a glucose challenge were several times higher than in obese children with low concentrations of the fatty acid as well as in lean controls. Declining and even insufficient insulin levels were observed in obese adolescents with high levels of the fatty acid. In isolated human islets exposed to palmitate we have observed insulin hypersecretion after 2 days exposure. In contrast, insulin secretion from the islets was reduced after 7 days culture in the presence of the fatty acid. This study aims at identifying islet-related biological events potentially linked with the observed insulin hypersecretion and later secretory decline in these obese children and adolescents using the islet model. We analyzed protein expression data obtained from human islets exposed to elevated palmitate levels for 2 and 7 days by an improved methodology for statistical analysis of differentially expressed proteins. Protein profiling of islet samples by liquid chromatography-tandem mass spectrometry identified 115 differentially expressed proteins (DEPs). Several DEPs including sorcin were associated with increased glucose-stimulated insulin secretion in islets after 2 days of exposure to palmitate. Similarly, several metabolic pathways including altered protein degradation, increased autophagy, altered redox condition, and hampered insulin processing were coupled to the functional impairment of islets after 7 days of culture in the presence of palmitate. Such biological events, once validated in the islets, may give rise to novel treatment strategies aiming at normalizing insulin levels in obese children with high palmitate levels, which may reduce or even prevent obesity-related type 2 diabetes mellitus.

  • 19.
    Hagman, Charlotte
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Ramström, Margareta
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Jansson, Maria
    James, Peter
    Håkansson, Per
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Reproducibility of Tryptic Digestion Investigated by Quantitative Fourier Transform Ion Cyclotron Resonance Mass Spectrometry2005In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 4, no 2, p. 394-399Article in journal (Refereed)
    Abstract [en]

    In this study, the reproducibility of tryptic digestion of complex solutions was investigated using liquid chromatography Fourier transform ion cyclotron resonance (LC FT-ICR) mass spectrometry. Tryptic peptides, from human cerebrospinal fluid, (CSF) was labeled with Quantification-Using-Enhanced-Signal-Tags (QUEST)-markers, or 1-((H4)nicotinoyloxy)- and 1-((D4)nicotinoyloxy)-succinimide ester markers. The analysis was performed on abundant proteins with respect-to-intensity ratios and sequence coverage and obtained by comparing differently labeled components from one or different pools. To interpret the dynamics in the proteome, one must be able to estimate the error introduced in each experimental steps. The intra sample variation due to derivatization was approximately 10%. The inter sample variation depending on derivatization and tryptic digestion was not more than approximately 30%. These experimental observations provide a range for the up- and down-regulations that are possible to study with electrospray ionization LC FT-ICR mass spectrometry.

  • 20. Hanisch, Katja
    et al.
    Soininen, Hilkka
    Alafuzoff, Irina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Hoffmann, Ralf
    Analysis of human tau in cerebrospinal fluid2010In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 9, no 3, p. 1476-1482Article in journal (Refereed)
    Abstract [en]

    Alzheimer's disease (AD) is the most common form of dementia. Neuropathologically, it is characterized by two major hallmarks: neurofibrillary tangles (NFT) formed from hyperphosphorylated versions of the tau-protein, and neuritic plaques (NP) containing mostly beta-amyloid peptides (A beta) that are formed from the amyloid precursor protein (APP) by enzymatic cleavage. Despite much progress in recent years, the causes of sporadic (i.e., nonfamiliar) AD are still unclear and its valid diagnosis still relies on autopsy. Clinically used biomarkers present in cerebrospinal fluid (CSF), that is, unphosphorylated or phosphorylated tau and A beta-peptides of different lengths, lack the necessary specificity and sensitivity. Here, we describe a novel strategy to characterize tau versions present in CSF with respect to their molecular mass and isoelectric point. Aliquots of 1 mL CSF (i.e., 700 to 1300 pg tau) from nondemented persons and histopathologically confirmed AD cases were depleted for six dominant proteins, separated by two-dimensional gel electrophoresis, and then electro-transferred onto PVDF-membranes. Tau was detected with monoclonal antibody (mAb) HT7 conjugated with horseradish peroxidase (HRP). In this way, a complex tau pattern was identified in CSF that was very similar to the tau preparations from autopsy brain samples. The presented strategy enables the analysis of the phosphorylation and processing status of tau in CSF samples from healthy people and patients diagnosed with different neurological disorders. This more-detailed information on circulating tau versions and their clearance rates may facilitate the development of new diagnostic tools.

  • 21.
    Hanrieder, Jörg
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
    Nyakas, Adrien
    Naessén, Tord
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
    Proteomic Analysis of Human Follicular Fluid Using an Alternative Bottom-Up Approach2008In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 7, no 1, p. 443-449Article in journal (Refereed)
    Abstract [en]

    Human follicular fluid (hFF) is the in vivo environment of oocytes during follicular maturation in the ovaries. It contains a huge variety of compounds such as, e.g., proteins that might play an important role in follicular development and oocyte growth. Previous proteomic studies on follicular fluid have isolated and already identified a certain number of proteins. Nevertheless, only a small part of proteins present in follicular fluid have been covered so far and a large number have still not been identified. Therefore, the need for new, more resolving, and sensitive approaches in proteome research is evident. We utilized a proteomic setup based on in solution isoelectric focusing (IEF) and reversed-phase nanoliquid chromatography coupled to matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (nano-LC MALDI TOF/TOF MS) for in depth protein analysis of human follicular fluid samples of patients undergoing controlled ovarian hyper stimulation (COH) for in vitro fertilization therapy (IVF). This approach led to the significant identification of 69 proteins, where 32 have not been reported before to be found in human follicular fluid with proteomic methods. Among these findings, at least two relevant compounds essentially involved in hormone secretion regulation during the folliculogenetic process were identified: sex hormone binding globulin (SHBG) and inhibin A (INHA). To confirm these results, both proteins were further validated by immunoassays.

  • 22. Hayes, Jerrard M
    et al.
    Frostell, Asa
    Cosgrave, Eoin F J
    Struwe, Weston B
    Potter, Oscar
    Davey, Gavin P
    Karlsson, Robert
    Anneren, Cecilia
    GE Healthcare, Rapsgatan 23, SE-75184 Uppsala, Sweden.
    Rudd, Pauline M
    Fc Gamma Receptor Glycosylation Modulates the Binding of IgG Glycoforms: A Requirement for Stable Antibody Interactions2014In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 13, no 12, p. 5471-5485Article in journal (Refereed)
    Abstract [en]

    FcγRs play a critical role in the immune response following recognition of invading particles and tumor associated antigens by circulating antibodies. In the present study we investigated the role of FcγR glycosylation in the IgG interaction and observed a stabilizing role for receptor N-glycans. We performed a complete glycan analysis of the recombinant FcγRs (FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIaPhe158/Val158, and FcγRIIIb) expressed in human cells and demonstrate that receptor glycosylation is complex and varied between receptors. We used surface plasmon resonance to establish binding patterns between rituximab and all receptors. Complex binding was observed for FcγRIa and FcγRIIIa. The IgG-FcγR interaction was further investigated using a combination of kinetic experiments and enzymatically deglycosylated FcγRIa and FcγRIIIaPhe158/Val158 receptors in an attempt to determine the underlying binding mechanism. We observed that antibody binding levels decreased for deglycosylated receptors, and at the same time, binding kinetics were altered and showed a more rapid approach to steady state, followed by an increase in the antibody dissociation rate. Binding of rituximab to deglycosylated FcγRIIIaPhe158 was now consistent with a 1:1 binding mechanism, while binding of rituximab to FcγRIIIaVal158 remained heterogeneous. Kinetic data support a complex binding mechanism, involving heterogeneity in both antibody and receptor, where fucosylated and afucosylated antibody forms compete in receptor binding and in receptor molecules where heterogeneity in receptor glycosylation plays an important role. The exact nature of receptor glycans involved in IgG binding remains unclear and determination of rate and affinity constants are challenging. Here, the use of more extended competition experiments appear promising and suggest that it may be possible to determine dissociation rate constants for high affinity afucosylated antibodies without the need to purify or express such variants. The data described provide further insight into the complexity of the IgG-FcγR interaction and the influence of FcγR glycosylation.

  • 23. 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.

  • 24.
    Karlsson, Oskar
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Environmental toxicology.
    Kultima, Kim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Wadensten, Henrik
    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.
    Roman, Erika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Andrén, Per E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Brittebo, Eva B.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Neurotoxin-Induced Neuropeptide Perturbations in Striatum of Neonatal Rats2013In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 12, no 4, p. 1678-1690Article in journal (Refereed)
    Abstract [en]

    The cyanobacterial toxin β-N-methylamino-l-alanine (BMAA) is suggested to play a role in neurodegenerative disease. We have previously shown that although the selective uptake of BMAA in the rodent neonatal striatum does not cause neuronal cell death, exposure during the neonatal development leads to cognitive impairments in adult rats. The aim of the present study was to characterize the changes in the striatal neuropeptide systems of male and female rat pups treated neonatally (postnatal days 9-10) with BMAA (40-460 mg/kg). The label-free quantification of the relative levels of endogenous neuropeptides using mass spectrometry revealed that 25 peptides from 13 neuropeptide precursors were significantly changed in the rat neonatal striatum. The exposure to noncytotoxic doses of BMAA induced a dose-dependent increase of neurosecretory protein VGF-derived peptides, and changes in the relative levels of cholecystokinin, chromogranin, secretogranin, MCH, somatostatin and cortistatin-derived peptides were observed at the highest dose. In addition, the results revealed a sex-dependent increase in the relative level of peptides derived from the proenkephalin-A and protachykinin-1 precursors, including substance P and neurokinin A, in female pups. Because several of these peptides play a critical role in the development and survival of neurons, the observed neuropeptide changes might be possible mediators of BMAA-induced behavioral changes. Moreover, some neuropeptide changes suggest potential sex-related differences in susceptibility toward this neurotoxin. The present study also suggests that neuropeptide profiling might provide a sensitive characterization of the BMAA-induced noncytotoxic effects on the developing brain.

  • 25.
    Kask, Lena
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Coagulation and inflammation science. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Jorsback, Anneli
    Winkvist, Maria
    Alfredsson, Jenny
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Coagulation and inflammation science.
    Ek, Bo
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Siegbahn, Agneta
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Coagulation and inflammation science.
    Identification of Novel Downstream Molecules of Tissue Factor Activation by Comparative Proteomic Analysis2014In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 13, no 2, p. 477-488Article in journal (Refereed)
    Abstract [en]

    Tissue factor (TF) is both an initiator of blood coagulation and a signaling receptor. Using a proteomic approach, we investigated the role of TF in cell signaling when stimulated by its ligand, activated factor VII (FVIIa). From a 2-D difference gel electrophoresis (DIGE) study we found forty one spots that were differentially regulated over time in FVIIa stimulated cells or in comparison to nonstimulated cells. Mass spectrometry identifies 23 out of these as 13 different proteins. One of them, elongation factor 2 (EF-2), was investigated in greater detail by Western blot, a protein synthesis assay and cell cycle analysis. When tissue factor was stimulated by FVIIa, the phosphorylation of EF-2 increased which inactivates this protein. Analyzing the effect using site inactivated FVIIa (FVIIai), as well as the protease activated receptor 2 (PAR-2) agonist SLIGKV, indicated that the inactivation was not PAR-2 dependent. A panel of tissue factor mutants was analyzed further to try to pinpoint what part of the cytoplasmic domain that is needed for this effect. Performing a protein synthesis assay in two different cell lines we could confirm that protein synthesis decreased upon stimulation by FVIIa. Cell cycle analysis showed that FVIIa also promotes a higher degree of cell proliferation.

  • 26.
    Kushnir, Mark M.
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Naessén, Tord
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health, Obstetrics and Gynaecology.
    Wånggren, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health, Obstetrics and Gynaecology.
    Rockwood, Alan L.
    Crockett, David K.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Protein and Steroid Profiles in Follicular Fluid after Ovarian Hyperstimulation as Potential Biomarkers of IVF Outcome2012In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 11, no 10, p. 5090-5100Article in journal (Refereed)
    Abstract [en]

    Controlled ovarian hyperstimulation is performed to assist with generation of multiple mature oocytes for use in in vitro fertilization (IVF). The goal of our study was to evaluate differences in protein and steroid profiles in ovarian follicular fluid (hFF) samples obtained during oocy-te retrieval from women undergoing IVF treatment and to identify physiological pathways associated with the proteins. The hFF samples were depleted of abundant proteins, fractionated by ultrafiltration, digested, and analyzed by nano-LC QTOF. Concentrations of 15 endogenous steroids were determined in the samples using LC-MS/MS methods. The total number of proteins identified in the samples was 75, of which 4, 7, and 2 were unique to the samples from women with viable pregnancy, miscarriage, and no pregnancy, respectively. Identified proteins were associated with the acute response signaling, coagulation system, intrinsic and extrinsic prothrombin activation, complement system, neuroprotective role of THOP1, FXR/RXR activation, role of tissue factor, and growth hormone pathways. A greater number of proteins associated with biosynthesis was found in hFF samples corresponding to the oocytes resulting in pregnancy. The abundance of seven proteins was found to be associated with steroidogenesis. The obtained data will contribute to better understanding of the pathogenesis and development of noninvasive markers for assessment of oocytes viability.

  • 27.
    Köcher, Thomas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Savitski, Mikhail M
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Nielsen, Michael L
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Zubarev, Roman A
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    PhosTShunter: A fast and reliable tool to detect phosphorylated peptides in liquid chromatography Fourier transform tandem mass spectrometry data sets2006In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 5, no 3, p. 659-668Article in journal (Refereed)
    Abstract [en]

    A database independent search algorithm for the detection of phosphopeptides is described. The program interrogates the tandem mass spectra of LC-MS/MS data sets regarding the presence of phosphorylation specific signatures. To achieve maximum informational content, the complementary fragmentation techniques electron capture dissociation (ECD) and collisionally activated dissociation (CAD) are used independently for peptide fragmentation. Several criteria characteristic for peptides phosphorylated on either serine or threonine residues were evaluated. The final algorithm searches for product ions generated by either the neutral loss of phosphoric acid or the combined neutral loss of phosphoric acid and water. Various peptide mixtures were used to evaluate the program. False positive results were not observed because the program utilizes the parts-per-million mass accuracy of Fourier transform ion cyclotron resonance mass spectrometry. Additionally, false negative results were not generated owing to the high sensitivity of the chosen criteria. The limitations of database dependent data interpretation tools are discussed and the potential of the novel algorithm to overcome these limitations is illustrated.

  • 28. Lalle, Marco
    et al.
    Camerini, Serena
    Cecchetti, Serena
    Sayadi, Ahmed
    Department of Biochemical Sciences, University of Rome “Sapienza”, Rome, Italy.
    Crescenzi, Marco
    Pozio, Edoardo
    Interaction network of the 14-3-3 protein in the ancient protozoan parasite Giardia duodenalis.2012In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 11, no 5, p. 2666-2683Article in journal (Refereed)
    Abstract [en]

    14-3-3s are phosphoserine/phosphotreonine binding proteins that play pivotal roles as regulators of multiple cellular processes in eukaryotes. The flagellated protozoan parasite Giardia duodenalis, the causing agent of giardiasis, is a valuable simplified eukaryotic model. A single 14-3-3 isoform (g14-3-3) is expressed in Giardia, and it is directly involved in the differentiation of the parasite into cyst. To define the overall functions of g14-3-3, the protein interactome has been investigated. A transgenic G. duodenalis strain was engineered to express a FLAG-tagged g14-3-3 under its own promoter. Affinity chromatography coupled with tandem mass spectrometry analysis have been used to purify and identify FLAG-g14-3-3-associated proteins from trophozoites and encysting parasites. A total of 314 putative g14-3-3 interaction partners were identified, including proteins involved in several pathways. Some interactions seemed to be peculiar of one specific stage, while others were shared among the different stages. Furthermore, the interaction of g14-3-3 with the giardial homologue of the CDC7 protein kinase (gCDC7) was characterized, leading to the identification of a multiprotein complex containing not only g14-3-3 and gCDC7 but also a newly identified and highly divergent homologue of DBF4, the putative regulatory subunit of gCDC7. The relevance of g14-3-3 interactions in G. duodenalis biology was discussed.

  • 29.
    Lindskog, Cecilia
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Kuhlwilm, Martin
    Davierwala, Armaity
    Fu, Ning
    Hedge, Geeta
    Uhlén, Mathias
    Navani, Sanjay
    Pääbo, Svante
    Pontén, Fredrik
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Analysis of Candidate Genes for Lineage-Specific Expression Changes in Humans and Primates2014In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 13, no 8, p. 3596-3606Article in journal (Refereed)
    Abstract [en]

    RUNX2, a gene involved in skeletal development, has previously been shown to be potentially affected by positive selection during recent human evolution. Here we have used antibody-based proteomics to characterize potential differences in expression patterns of RUNX2 interacting partners during primate evolution. Tissue microarrays consisting of a large set of normal tissues from human and macaque were used for protein profiling of 50 RUNX2 partners with immunohistochemistry. Eleven proteins (AR, CREBBP, EP300, FGF2, HDAC3, JUN, PRKD3, RUNX1, SATB2, TCF3, and YAP1) showed differences in expression between humans and macaques. These proteins were further profiled in tissues from chimpanzee, gorilla, and orangutan, and the corresponding genes were analyzed with regard to genomic features. Moreover, protein expression data were compared with previously obtained RNA sequencing data from six different organs. One gene (TCF3) showed significant expression differences between human and macaque at both the protein and RNA level, with higher expression in a subset of germ cells in human testis compared with macaque. In conclusion, normal tissues from macaque and human showed differences in expression of some RUNX2 partners that could be mapped to various defined cell types. The applied strategy appears advantageous to characterize the consequences of altered genes selected during evolution.

  • 30. Mardinoglu, Adil
    et al.
    Kampf, Caroline
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Asplund, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Fagerberg, Linn
    Hallstrom, Bjorn M.
    Edlund, Karolina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Blueher, Matthias
    Pontén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Uhlen, Mathias
    Nielsen, Jens
    Defining the Human Adipose Tissue Proteome To Reveal Metabolic Alterations in Obesity2014In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 13, no 11, p. 5106-5119Article in journal (Refereed)
    Abstract [en]

    White adipose tissue (WAT) has a major role in the progression of obesity. Here, we combined data from RNA-Seq and antibody-based immunohistochemistry to describe the normal physiology of human WAT obtained from three female subjects and explored WAT-specific genes by comparing WAT to 26 other major human tissues. Using the protein evidence in WAT, we validated the content of a genome-scale metabolic model for adipocytes. We employed this high-quality model for the analysis of subcutaneous adipose tissue (SAT) gene expression data obtained from subjects included in the Swedish Obese Subjects Sib Pair study to reveal molecular differences between lean and obese individuals. We integrated SAT gene expression and plasma metabolomics data, investigated the contribution of the metabolic differences in the mitochondria of SAT to the occurrence of obesity, and eventually identified cytosolic branched-chain amino acid (BCAA) transaminase 1 as a potential target that can be used for drug development. We observed decreased glutaminolysis and alterations in the BCAAs metabolism in SAT of obese subjects compared to lean subjects. We also provided mechanistic explanations for the changes in the plasma level of BCAAs, glutamate, pyruvate, and alpha-ketoglutarate in obese subjects. Finally, we validated a subset of our model-based predictions in 20 SAT samples obtained from 10 lean and 10 obese male and female subjects.

  • 31. Marin-Vicente, Consuelo
    et al.
    Guerrero-Valero, Marta
    Nielsen, Michael L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Savitski, Mikhail M.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Gomez-Fernandez, Juan C.
    Zubarev, Roman A.
    Corbalan-Garcia, Senena
    ATP Enhances Neuronal Differentiation of PC12 Cells by Activating PKC alpha Interactions with Cytoskeletal Proteins2011In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 10, no 2, p. 529-540Article in journal (Refereed)
    Abstract [en]

    PKC alpha is a key mediator of the neuronal differentiation controlled by NGF and ATP. However, its down stream signaling pathways remain to be elucidated. To identify the signaling partners of PKC alpha, we analyzed proteins coimmunoprecipitated with this enzyme in PC12 cells differentiated with NGF and ATP and compared them with those obtained with NGF alone or growing media. Mass spectrometry analysis (LC-MS/MS) identified plectin, peripherin, filamin A, fascin, and beta-actin as potential interacting proteins. The colocalization of PKC alpha and its interacting proteins increased when PC12 cells were differentiated with NGF and ATP. Peripherin and plectin organization and the cortical remodeling of beta-actin were dramatically affected when PKC alpha was down-regulated, suggesting that all three proteins might be functional targets of ATP-dependent PKC alpha signaling. Taken together, these data demonstrate that PKC alpha is essential for controlling the neuronal development induced by NGF and ATP and interacts with the cytoskeletal components at two levels: assembly of the intermediate filament peripherin and organization of cortical actin.

  • 32.
    Musunuri, Sravani
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Wetterhall, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Ingelsson, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Artemenko, Konstantin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Kultima, Kim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Shevchenko, Ganna
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Quantification of the Brain Proteome in Alzheimer's Disease Using Multiplexed Mass Spectrometry2014In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 13, no 4, p. 2056-2068Article in journal (Refereed)
    Abstract [en]

    We have compared the brain proteome in the temporal neocortex between Alzheimer's disease (AD) patients and non-AD individuals by using shotgun mass spectrometry based on a stable isotope dimethyl labeling. A total of 827 unique proteins were identified and quantitated. Of these, 227 proteins were found in at least 9 out of 10 AD/control pairs and were further subjected to statistical analysis. A total of 69 proteins showed different levels (p-value < 0.05) in AD versus control brain samples. Of these proteins, 37 were increased and 32 were decreased as compared to the non-AD subjects. Twenty-three proteins comprise novel proteins that have not previously been reported as related to AD, e.g., neuronal-specific septin-3, septin-2, septin-5, dihydropteridine reductase, and clathrin heavy chain 1. The proteins with altered levels in the AD brain represent a wide variety of pathways suggested to be involved in the disease pathogenesis, including energy metabolism, glycolysis, oxidative stress, apoptosis, signal transduction, and synaptic functioning. Apart from leading to new insights into the molecular mechanisms in AD, the findings provide us with possible novel candidates for future diagnostic and prognostic disease markers.

  • 33.
    Nilsson, Anna
    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.
    Sjögren, Benita
    Svensson, Marcus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Pierson, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Zhang, Xiaoqun
    Caprioli, Richard M.
    Buijs, Jos
    Persson, Björn
    Svenningsson, Per
    Andrén, Per E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Increased striatal mRNA and protein levels of the immunophilin FKBP-12 in experimental Parkinson’s Disease and identification of FKBP-12-binding proteins2007In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 6, no 10, p. 3952-3961Article in journal (Refereed)
    Abstract [en]

    FKBP-12, a 12 kDa FK506-binding protein (neuroimmunophilin), acts as a receptor for the immunosuppressant drug FK506. Neuroimmunophilins, including FKBP-12, are abundant in the brain and have been shown to be involved in reversing neuronal degeneration and preventing cell death. In this report, we have utilized several analytical techniques, such as in situ hybridization, Western blotting, two-dimensional gel electrophoresis, and liquid chromatography electrospray tandem mass spectrometry to study the transcriptional expression as well as protein levels of FKBP-12 in the unilateral 6-hydroxydopamine (6-OHDA) rat model of Parkinson's disease. The FKBP-12 protein was also detected directly on brain tissue sections using mass spectrometry profiling. We found increased levels of FKBP-12 mRNA and protein in the dorsal and middle part of the 6-OHDA lesioned striatum. Thus, these studies clearly demonstrate that FKBP-12 is increased in the brain of a common animal model of Parkinson's disease (PD). Additionally, we have identified potential binding partners to FKBP-12 that may be implicated in the pathophysiology of Parkinson's disease, such as alpha-enolase, 14-3-3 zeta/delta, pyruvate kinase isozymes, and heat shock protein 70, using surface plasmon resonance sensor technology in combination with mass spectrometry. In conclusion, these data strongly suggests that FKBP-12 is altered in an experimental model of PD.

  • 34. Nilsson, Carol L.
    et al.
    Berven, Frode
    Selheim, Frode
    Liu, Huiling
    Moskal, Joseph R.
    Kroes, Roger A.
    Sulman, Erik P.
    Conrad, Charles A.
    Lang, Frederick F.
    Andren, Per E.
    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.
    Carlsohn, Elisabet
    Lilja, Hans
    Malm, Johan
    Fenyoe, David
    Subramaniyam, Devipriya
    Wang, Xiangdong
    Gonzales-Gonzales, Maria
    Dasilva, Noelia
    Diez, Paula
    Fuentes, Manuel
    Vegvari, Akos
    Sjodin, Karin
    Welinder, Charlotte
    Laurell, Thomas
    Fehniger, Thomas E.
    Lindberg, Henrik
    Rezeli, Melinda
    Edula, Goutham
    Hober, Sophia
    Marko-Varga, Gyorgy
    Chromosome 19 Annotations with Disease Speciation: A First Report from the Global Research Consortium2013In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 12, no 1, p. 134-149Article in journal (Refereed)
    Abstract [en]

    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.

  • 35. Nilsson, Carol L.
    et al.
    Mostovenko, Ekaterina
    Lichti, Cheryl F.
    Ruggles, Kelly
    Fenyoe, David
    Rosenbloom, Kate R.
    Hancock, William S.
    Paik, Young-Ki
    Omenn, Gilbert S.
    LaBaer, Joshua
    Kroes, Roger A.
    Uhlen, Matthias
    Hober, Sophia
    Vegvari, Akos
    Andrén, Per E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Sulman, Erik P.
    Lang, Frederick F.
    Fuentes, Manuel
    Carlsohn, Elisabet
    Emmett, Mark R.
    Moskal, Joseph R.
    Berven, Frode S.
    Fehniger, Thomas E.
    Marko-Varga, Gyorgy
    Use of ENCODE Resources to Characterize Novel Proteoforms and Missing Proteins in the Human Proteome2015In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 14, no 2, p. 603-608Article in journal (Refereed)
    Abstract [en]

    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.

  • 36.
    Nilsson, Stefan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Ramström, Margareta
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Palmblad, Magnus
    The Ångström Laboratory, Division of Ion Physics, Uppsala University.
    Axelsson, Ove
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health, Obstetrics and Gynaecology.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Explorative study of the protein composition of amniotic fluid by liquid chromatography electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry2004In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 3, no 4, p. 884-889Article in journal (Other academic)
    Abstract [en]

    To explore the suitability of FTICR mass spectrometry for the analysis of the protein composition of amniotic fluid (AF), an AF sample from 15 weeks gestation from a healthy 36-year-old woman was tryptically digested, with or without prior serum albumin removal. The tryptic peptides were separated by gradient capillary liquid chromatography (LC) followed by electrospray ionization (ESI) and mass spectrometric detection with a 9.4 T Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR). The obtained data underwent computer-aided mathematical and statistical evaluation to extract significant tryptic peptide patterns from human proteins. Forty-three proteins were putatively identified; among them were known protein constituents of amniotic fluid, but also many that not have been detected before. The removal of serum albumin prior to tryptic digestion reduced ion suppression from abundant HSA fragments. The protein analysis of amniotic fluid by albumin removal, tryptic digestion and LC/FT-ICR-MS analysis was found to be a straightforward technique.

  • 37.
    Nyblom, Hanna K
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Bugliani, Marco
    Fung, Eva
    Boggi, Ugo
    Zubarev, Roman
    Marchetti, Piero
    Bergsten, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Apoptotic, Regenerative, And Immune-Related Signaling in Human Islets from Type 2 Diabetes Individuals2009In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 8, no 12, p. 5650-5656Article in journal (Refereed)
    Abstract [en]

    Islet dysfunction is a primary cause of developing type 2 diabetes mellitus (T2DM). Events leading to islet failure are still poorly defined due to the complexity of the disease and scarcity of human T2DM islets. The aim of the present study was to identify cellular mechanisms involved in the T2DM pathophysiology by protein profiling islets obtained from T2DM individuals and age- and weight-matched controls using liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry and surface enhanced laser desorption/ionization time-of-flight mass spectrometry. In T2DM islets, multiple differentially expressed proteins correlated with insulin secretion. When these T2DM islet proteins were analyzed for differential pathway activation, three of the five most activated pathways were pathways of cell arrest and apoptosis (p53, caspase, stress-activated), one represented immune-response (Fas), and the most activated pathway was connected with proliferation and regeneration (E2F). Among the inactivated pathways, three out of five were pathways of proliferation and regeneration (insulin, PRL, PDGF). The present study is the first to report differential activation of specific pathways during T2DM islet deterioration. The information about alterations in pathway signaling patterns may open new ways to develop strategies aimed at restoring islet cell function and survival.

  • 38.
    Ow, Saw Yen
    et al.
    Department of Chemical and Process Engineering, The University of Sheffield.
    Cardona, Tanai
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Molecular Biomimetics.
    Taton, Arnaud
    Virginia Commonwealth University.
    Magnuson, Ann
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Molecular Biomimetics.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Stensjö, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Wright, Philip C
    Department of Chemical and Process Engineering, The University of Sheffield.
    Quantitative shotgun proteomics of enriched heterocysts from Nostoc sp. PCC 7120 using 8-Plex isobaric peptide tags2008In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 7, no 4, p. 1615-1628Article in journal (Refereed)
    Abstract [en]

    The filamentous cyanobacterium Nostoc sp. strain PCC 7120 is capable of fixing atmospheric nitrogen. The labile nature of the core process requires the terminal differentiation of vegetative cells to form heterocysts, specialized cells with altered cellular and metabolic infrastructure to mediate the N2-fixing process. We present an investigation targeting the cellular proteomic expression of the heterocysts compared to vegetative cells of a population cultured under N2-fixing conditions. New 8-plex iTRAQ reagents were used on enriched replicate heterocyst and vegetative cells, and replicate N2-fixing and non-N2-fixing filaments to achieve accurate measurements. With this approach, we successfully identified 506 proteins, where 402 had confident quantifications. Observations provided by purified heterocyst analysis enabled the elucidation of the dominant metabolic processes between the respective cell types, while emphasis on the filaments enabled an overall comparison. The level of analysis provided by this investigation presents various tools and knowledge that are important for future development of cyanobacterial biohydrogen production.

  • 39.
    Ow, Saw Yen
    et al.
    Department of Chemical and Process Engineering, The University of Sheffield.
    Noirel, Josselin
    Department of Chemical and Process Engineering, The University of Sheffield.
    Cardona, Tanai
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Molecular Biomimetics.
    Taton, Arnaud
    Virginia Commonwealth University.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Stensjö, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Microbial Chemistry.
    Wright, Philip C
    Department of Chemical and Process Engineering, The University of Sheffield.
    Quantitative overview of N2 fixation in Nostoc punctiforme ATCC 29133 through cellular enrichments and iTRAQ shotgun proteomics2009In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 8, no 1, p. 187-198Article in journal (Refereed)
    Abstract [en]

    Nostoc punctiforme ATCC 29133 is a photoautotrophic cyanobacterium with the capacity to fix atmospheric N2. Its ability to mediate this process is similar to that described for Nostoc sp. PCC 7120, where vegetative cells differentiate into heterocysts. Quantitative proteomic investigations at both the filament level and the heterocyst level are presented using isobaric tagging technology (iTRAQ), with 721 proteins at the 95% confidence interval quantified across both studies. Observations from both experiments yielded findings confirmatory of both transcriptional studies, and published Nostoc sp. PCC 7120 iTRAQ data. N. punctiforme exhibits similar metabolic trends, though changes in a number of metabolic pathways are less pronounced than in Nostoc sp. PCC 7120. Results also suggest a number of proteins that may benefit from future investigations. These include ATP dependent Zn-proteases, N-reserve degraders and also redox balance proteins. Complementary proteomic data sets from both organisms present key precursor knowledge that is important for future cyanobacterial biohydrogen research.

  • 40. 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.

  • 41.
    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.

  • 42.
    Ramström, Margareta
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Hagman, Charlotte
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Mitchell, Jennifer
    Derrick, Peter
    Håkansson, Per
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Depletion of High-Abundant Proteins in Body Fluids Prior to Liquid Chromatography Fourier Transform Ion Cyclotron Resonance Mass Spectrometry2005In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 4, no 2, p. 410-416Article in journal (Refereed)
    Abstract [en]

    Today, proteomics is an exciting approach to discover potential biomarkers of different disorders. One challenge with proteomics experiments is the wide concentration range of proteins in various tissues and body fluids. The most abundant component in human body fluids, human serum albumin (HSA), is present at concentrations corresponding to approximately 50% of the total protein content in e.g., plasma and cerebrospinal fluid (CSF). If this component could be selectively removed, then the chances of observing lower-abundance component of clinical interest would be greatly improved. There are today several approaches of varying specificity available for depletion. In this study, the properties of two commercially available kits, for the removal of HSA and HSA and immunoglobulin G (lgG), respectively, were compared, and the benefits of using depletion steps prior to on-line LC-FTICR MS were evaluated. Both methods were applied on plasma and CSF. To our knowledge, these are the first results reported for CSF. Also, the combination with electrospray LC-FTICR MS is novel. The proportion of depleted HSA and lgG was estimated using global labeling markers for peptide quantification. Both depletion-methods provided a significant reduction of HSA, and the identification of lower abundant components was clearly facilitated. A higher proportion of HSA was removed using the affinity-based removal kit, and consequently more proteins could be identified using this approach.

  • 43.
    Rezeli, Melinda
    et al.
    Lund Univ, Clin Prot Sci & Imaging, Dept Biomed Engn, BMC D13, SE-22184 Lund, Sweden..
    Sjödin, Karin
    H Lundbeck & Co AS, Dept Drug Metab, DK-2500 Copenhagen, Denmark..
    Lindberg, Henrik
    Lund Univ, Clin Prot Sci & Imaging, Dept Biomed Engn, BMC D13, SE-22184 Lund, Sweden..
    Gidlöf, Olof
    Lund Univ, Skane Univ Hosp, Dept Cardiol, Clin Sci, SE-22185 Lund, Sweden..
    Lindahl, Bertil
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cardiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, UCR-Uppsala Clinical Research Center.
    Jernberg, Tomas
    Karolinska Inst, Danderyd Univ Hosp, Dept Clin Sci, SE-18288 Stockholm, Sweden..
    Spaak, Jonas
    Karolinska Inst, Danderyd Univ Hosp, Dept Clin Sci, SE-18288 Stockholm, Sweden..
    Erlinge, David
    Lund Univ, Skane Univ Hosp, Dept Cardiol, Clin Sci, SE-22185 Lund, Sweden..
    Marko-Varga, György
    Lund Univ, Clin Prot Sci & Imaging, Dept Biomed Engn, BMC D13, SE-22184 Lund, Sweden..
    Quantitation of 87 Proteins by nLC-MRM/MS in Human Plasma: Workflow for Large-Scale Analysis of Biobank Samples2017In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 16, no 9, p. 3242-3254Article in journal (Refereed)
    Abstract [en]

    A multiple reaction monitoring (MRM) assay was developed for precise quantitation of 87 plasma proteins including the three isoforms of apolipoprotein E (APOE) associated with cardiovascular diseases using nanoscale liquid chromatography separation and stable isotope dilution strategy. The analytical performance of the assay was evaluated and we found an average technical variation of 4.7% in 4-5 orders of magnitude dynamic range (approximate to 0.2 mg/L to 4.5 g/L) from whole plasma digest. Here, we report a complete workflow, including sample processing adapted to 96-well plate format and normalization strategy for large-scale studies. To further investigate the MS-based quantitation the amount of six selected proteins was measured by routinely used clinical chemistry assays as well and the two methods showed excellent correlation with high significance (p-value < 10e-5) for the six proteins, in addition for the cardiovascular predictor factor, APOB: APOA1 ratio (r = 0.969, p-value < 10e-5). Moreover, we utilized the developed assay for screening of biobank samples from patients with myocardial infarction and performed the comparative analysis of patient groups with STEMI (ST- segment elevation myocardial infarction), NSTEMI (non ST- segment elevation myocardial infarction) and type-2 AMI (type-2 myocardial infarction) patients.

  • 44. Rodriguez, Henry
    et al.
    Snyder, Mike
    Uhlén, Mathias
    Andrews, Phil
    Beavis, Ronald
    Borchers, Christoph
    Chalkley, Robert J
    Cho, Sang Yun
    Cottingham, Katie
    Dunn, Michael
    Dylag, Tomasz
    Edgar, Ron
    Hare, Peter
    Heck, Albert J R
    Hirsch, Roland F
    Kennedy, Karen
    Kolar, Patrik
    Kraus, Hans-Joachim
    Mallick, Parag
    Nesvizhskii, Alexey
    Ping, Peipei
    Pontén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Yang, Liming
    Yates, John R
    Stein, Stephen E
    Hermjakob, Henning
    Kinsinger, Christopher R
    Apweiler, Rolf
    Recommendations from the 2008 International Summit on Proteomics Data Release and Sharing Policy: the Amsterdam principles.2009In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 8, no 7, p. 3689-3692Article in journal (Refereed)
    Abstract [en]

    Policies supporting the rapid and open sharing of genomic data have directly fueled the accelerated pace of discovery in large-scale genomics research. The proteomics community is starting to implement analogous policies and infrastructure for making large-scale proteomics data widely available on a precompetitive basis. On August 14, 2008, the National Cancer Institute (NCI) convened the "International Summit on Proteomics Data Release and Sharing Policy" in Amsterdam, The Netherlands, to identify and address potential roadblocks to rapid and open access to data. The six principles agreed upon by key stakeholders at the summit addressed issues surrounding (1) timing, (2) comprehensiveness, (3) format, (4) deposition to repositories, (5) quality metrics, and (6) responsibility for proteomics data release. This summit report explores various approaches to develop a framework of data release and sharing principles that will most effectively fulfill the needs of the funding agencies and the research community.

  • 45. Rosenling, Therese
    et al.
    Slim, Christiaan L
    Christin, Christin
    Coulier, Leon
    Shi, Shanna
    Stoop, Marcel P
    Bosman, Jan
    Suits, Frank
    Horvatovich, Peter L
    Stockhofe-Zurwieden, Norbert
    Vreeken, Rob
    Hankemeier, Thomas
    van Gool, Alain J
    Luider, Theo M
    Bischoff, Rainer
    The effect of preanalytical factors on stability of the proteome and selected metabolites in cerebrospinal fluid (CSF).2009In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 8, no 12, p. 5511-22Article in journal (Refereed)
    Abstract [en]

    To standardize the use of cerebrospinal fluid (CSF) for biomarker research, a set of stability studies have been performed on porcine samples to investigate the influence of common sample handling procedures on proteins, peptides, metabolites and free amino acids. This study focuses at the effect on proteins and peptides, analyzed by applying label-free quantitation using microfluidics nanoscale liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (chipLC-MS) as well as matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT-ICR-MS) and Orbitrap LC-MS/MS to trypsin-digested CSF samples. The factors assessed were a 30 or 120 min time delay at room temperature before storage at -80 degrees C after the collection of CSF in order to mimic potential delays in the clinic (delayed storage), storage at 4 degrees C after trypsin digestion to mimic the time that samples remain in the cooled autosampler of the analyzer, and repeated freeze-thaw cycles to mimic storage and handling procedures in the laboratory. The delayed storage factor was also analyzed by gas chromatography mass spectrometry (GC-MS) and liquid chromatography mass spectrometry (LC-MS) for changes of metabolites and free amino acids, respectively. Our results show that repeated freeze/thawing introduced changes in transthyretin peptide levels. The trypsin digested samples left at 4 degrees C in the autosampler showed a time-dependent decrease of peak areas for peptides from prostaglandin D-synthase and serotransferrin. Delayed storage of CSF led to changes in prostaglandin D-synthase derived peptides as well as to increased levels of certain amino acids and metabolites. The changes of metabolites, amino acids and proteins in the delayed storage study appear to be related to remaining white blood cells. Our recommendations are to centrifuge CSF samples immediately after collection to remove white blood cells, aliquot, and then snap-freeze the supernatant in liquid nitrogen for storage at -80 degrees C. Preferably samples should not be left in the autosampler for more than 24 h and freeze/thaw cycles should be avoided if at all possible.

  • 46. Rosenling, Therese
    et al.
    Stoop, Marcel
    Attali, Amos
    van Aken, Hans
    Suidgeest, Ernst
    Christin, Christin
    Stingl, Christoph
    Suits, Frank
    Horvatovich, Peter
    Hintzen, Rogier
    Tuinstra, Tinka
    Bischoff, Rainer
    Luider, Theo
    Profiling and Identification of Cerebrospinal Fluid Proteins in a Rat EAE Model of Multiple Sclerosis.2012In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 11, no 4, p. 2048-2060Article in journal (Refereed)
    Abstract [en]

    The experimental autoimmune encephalomyelitis (EAE) model resembles certain aspects of multiple sclerosis (MScl), with common features such as motor dysfunction, axonal degradation, and infiltration of T-cells. We studied the cerebrospinal fluid (CSF) proteome in the EAE rat model to identify proteomic changes relevant for MScl disease pathology. EAE was induced in male Lewis rats by injection of myelin basic protein (MBP) together with complete Freund's adjuvant (CFA). An inflammatory control group was injected with CFA alone, and a nontreated group served as healthy control. CSF was collected at day 10 and 14 after immunization and analyzed by bottom-up proteomics on Orbitrap LC-MS and QTOF LC-MS platforms in two independent laboratories. By combining results, 44 proteins were discovered to be significantly increased in EAE animals compared to both control groups, 25 of which have not been mentioned in relation to the EAE model before. Lysozyme C1, fetuin B, T-kininogen, serum paraoxonase/arylesterase 1, glutathione peroxidase 3, complement C3, and afamin are among the proteins significantly elevated in this rat EAE model. Two proteins, afamin and complement C3, were validated in an independent sample set using quantitative selected reaction monitoring mass spectrometry. The molecular weights of the identified differentially abundant proteins indicated an increased transport across the blood-brain barrier (BBB) at the peak of the disease, caused by an increase in BBB permeability.

  • 47.
    Rossbach, Uwe
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Biological Research on Drug Dependence.
    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.
    Kultima, Kim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Zhou, Qin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Biological Research on Drug Dependence.
    Hallberg, Mathias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Biological Research on Drug Dependence.
    Gordh, Torsten
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Andren, Per E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Nyberg, Fred
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Biological Research on Drug Dependence.
    A quantitative peptidomic analysis of peptides related to the endogenous opioid and tachykinin systems in nucleus accumbens of rats following naloxone-precipitated morphine withdrawal2009In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 8, no 2, p. 1091-1098Article in journal (Refereed)
    Abstract [en]

    We have applied a recently developed label-free mass spectrometry based peptidomic approach to identify and quantify a variety of endogenous peptides from rat nucleus accumbens following withdrawal in naloxone-precipitated, morphine-dependent rats of two separate strains. We focused on maturated, partially processed and truncated peptides derived from the peptide precursors proenkephalin, prodynorphin and preprotachykinin. The expression of several identified peptides was dependent on strain and was affected during morphine withdrawal.

  • 48.
    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.

  • 49.
    Savitski, Mikhail M.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences.
    Nielsen, Michael L.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences.
    Kjeldsen, Frank
    Zubarev, Roman A.
    Proteomics-grade De Novo sequencing approach2005In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 4, no 6, p. 2348-2354Article in journal (Refereed)
    Abstract [en]

    The conventional approach in modern proteomics to identify proteins from limited information provided by molecular and fragment masses of their enzymatic degradation products carries an inherent risk of both false positive and false negative identifications. For reliable identification of even known proteins, complete de novo sequencing of their peptides is desired. The main problems of conventional sequencing based on tandem mass spectrometry are incomplete backbone fragmentation and the frequent overlap of fragment masses. In this work, the first proteomics-grade de novo approach is presented, where the above problems are alleviated by the use of complementary fragmentation techniques CAD and ECD. Implementation of a high-current, large-area dispenser cathode as a source of low-energy electrons provided efficient ECD of doubly charged peptides, the most abundant species (65-80%), in a typical trypsin-based proteomics experiment. A new linear de novo algorithm is developed combining efficiency and speed, processing on a conventional 3 GHz PC, 1000 MS/MS data sets in 60 s. More than 6% of all MS/MS data for doubly charged peptides yielded complete sequences, and another 13% gave nearly complete sequences with a maximum gap of two amino acid residues. These figures are comparable with the typical success rates (5-15%) of database identification. For peptides reliably found in the database (Mowse score > or = 34), the agreement with de novo-derived full sequences was >95%. Full sequences were derived in 67% of the cases when full sequence information was present in MS/MS spectra. Thus the new de novo sequencing approach reached the same level of efficiency and reliability as conventional database-identification strategies.

  • 50.
    Shevchenko, Ganna
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Musunuri, Sravani
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Wetterhall, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Comparison of Extraction Methods for the Comprehensive Analysis of Mouse Brain Proteome using Shotgun-based Mass Spectrometry2012In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 11, no 4, p. 2441-2451Article in journal (Refereed)
    Abstract [en]

    This study compares 16 different extraction methods for the comprehensive extraction of mouse brain proteome in combination with "shotgun"-based mass spectrometry (MS). Membrane proteins (MPs) are responsible for a large part of the regulatory functions of the cell and are therefore of great interest to extract and analyze. Sixteen protein extraction protocols were evaluated in regards to protein yield and number of identified proteins with emphasis on MPs. The extracted proteins were delipidated, on-filter digested, and analyzed by reversed phase nanoliquid chromatography (RP-nanoLC) in combination with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using a 7 T hybrid LTQ: FT mass spectrometer. Detergent-based lysis buffers showed higher efficiencies and yields in the extraction of proteins from the brain tissue compared to solubilization with organic solvents or organic acids. The detergent octyl-beta-D-glucopyranoside gave the highest number of identified proteins (541) as well as numbers and percentages of identified MPs (29%). Detergent-based protocols are the best sample preparation tools for central nervous system (CNS) tissue and can readily be applied to screen for candidate biomarkers of neurological diseases.

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