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  • 51.
    Lundberg, Emma
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Creation of an antibody-based subcellular protein atlas2010In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 10, no 22, p. 3984-3996Article, review/survey (Refereed)
    Abstract [en]

    An important part for understanding the complex machinery of living cells is to know the spatial distribution of proteins all the way from organ to organelle levels. An equally important part of proteomics is to map the subcellular distribution of all human proteins. Here, we discuss methodologies for systematic subcellular profiling with emphasis on the antibody-based approach performed as a part of the Human Protein Atlas project. The considerations made when creating the subcellular protein atlas and critical parameters of this approach are discussed.

  • 52.
    Ma, Wei-Juan
    et al.
    Medical College of Xi'an Jiaotong University, Key Laboratory of Environment and Genes Related to Diseases, Xi'an, Shaanxi, China.
    Guo, Xiong
    Medical College of Xi'an Jiaotong University, Key Laboratory of Environment and Genes Related to Diseases, Xi'an, Shaanxi, China.
    Liu, Jiang-Tao
    Medical College of Xi'an Jiaotong University, Key Laboratory of Environment and Genes Related to Diseases, Xi'an, Shaanxi, China.
    Liu, Rui-Yu
    Medical College of Xi'an Jiaotong University, Key Laboratory of Environment and Genes Related to Diseases, Xi'an, Shaanxi, China.
    Hu, Jian-Wen
    Research Center for Proteome Analysis, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China.
    Sun, An-Guo
    Research Center for Proteome Analysis, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China.
    Yu, Yue-Xiang
    Shaanxi Provincial Institute For Endemic Disease Control, Xi'an, Shaanxi, China.
    Lammi, Mikko
    Department of Biosciences, University of Eastern Finland, Kuopio, Finland.
    Proteomic changes in articular cartilage of human endemic osteoarthritis in China.2011In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 11, no 14, p. 2881-90, article id 21681992Article in journal (Refereed)
    Abstract [en]

    Kashin-Beck disease (KBD) is a chronic endemic osteochondropathy with unclear pathogenesis. It is a degenerative disease similar to osteoarthritis, but with different manifestations of cartilage damage. The aim of this investigation was to show the protein changes in KBD cartilage and to identify the candidate proteins in order to understand the pathogenesis of the disease. Proteins were extracted from the media of primary cell cultures of KBD and normal chondrocytes, and separated by two-dimensional fluorescence difference gel electrophoresis (2-D DIGE). MALDI-TOF/TOF analysis revealed statistically significant differences in 27 proteins from KBD chondrocyte cultures, which consisted of 17 up-regulated and ten down-regulated proteins. The results were further validated by Western blot analysis. The proteins identified are mainly involved in cellular redox homeostasis and stress response (MnSOD, Hsp27, Peroxiredoxin-1, and Cofilin-1), glycolysis (PGK-1, PGM-1, α-enolase), and cell motility and cytoskeletal organization (Actin, Calponin-2, and Keratin). These KBD-associated proteins indicate that cytoskeletal remodeling, glycometabolism, and oxidative stress are abnormal in KBD articular cartilage.

  • 53.
    Maddalo, Gianluca
    et al.
    Stockholm University, Faculty of Science, Department of Analytical Chemistry.
    Chovanec, Peter
    Stenberg-Bruzell, Filippa
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nielsen, Hailyn V
    Jensen-Seaman, Michael
    Ilag, Leopold
    Stockholm University, Faculty of Science, Department of Analytical Chemistry.
    Kline, Kimberly
    Daley, Daniel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    A reference map of the membrane proteome of Enterococcus faecalis2011In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 11, no Special issue, p. 3935-3941Article in journal (Refereed)
    Abstract [en]

    Enterococcus faecalis is a gram-positive bacterium that is part of the indigenous microbiotica of humans and animals as well as an opportunistic pathogen. In this study, we have fractionated the membrane proteome of E. faecalis and identified many of its constituents by mass spectrometry. We present blue native-/SDS-PAGE reference maps that contain 102 proteins. These proteins are important for cellular homeostasis, virulence, and antibiotic intervention. Intriguingly, many proteins with no known function were also identified, indicating that there are substantial gaps in the knowledge of this organism's biology. On a more limited scale, we also provide insight into the composition of membrane protein complexes. This study is a first step toward elucidating the membrane proteome of E. faecalis, which is critical for a better understanding of how this bacterium interacts with a host and with the extracellular milieu.

  • 54.
    Mi, Jia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Kirchner, Eva
    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.
    Quantitative proteomic comparison of mouse peroxisomes from liver and kidney2007In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 7, no 11, p. 1916-1928Article in journal (Refereed)
    Abstract [en]

    The peroxisome plays a central role in the catabolic and anabolic pathways that contribute to the lipid homeostasis. Besides this main function, this organelle has gained functional diversity. Although several approaches have been used for peroxisomal proteome analysis, a quantitative protein expression analysis of peroxisomes from different tissues has not been elucidated yet. Here, we applied a 2-DE-based method on mouse liver and kidney peroxisomal enriched fractions to study the tissue-dependent protein expression. Ninety-one spots were identified from the 2-DE maps from pH 3.0-10.0 and 51 spots from the basic range corresponding to 31 peroxisomal proteins, 10 putative peroxisomal, 6 cytosolic, 17 mitochondrial and 1 protein from endoplasmic reticulum. Based on the identification and on the equivalent quality of both tissue preparations, the differences emerging from the comparison could be quantified. In liver, proteins involved in pathways such as α- and β-oxidation, isoprenoid biosynthesis, amino acid metabolism and purine and pirimidine metabolism were more abundant whereas in kidney, proteins from the straight-chain fatty acid β-oxidation were highly expressed. These results indicate that tissue-specific functional classes of peroxisomal proteins could be relevant to study peroxisomal cellular responses or pathologies. Finally, a web-based peroxisomal proteomic database was built.

  • 55.
    Moruz, Luminita
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Käll, Lukas
    GradientOptimizer: An open-source graphical environment for calculating optimized gradients in reversed-phase liquid chromatography2014In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 14, no 12, p. 1464-1466Article in journal (Refereed)
    Abstract [en]

    We here present GradientOptimizer, an intuitive, lightweight graphical user interface to design nonlinear gradients for separation of peptides by reversed-phase liquid chromatography. The software allows to calculate three types of nonlinear gradients, each of them optimizing a certain retention time distribution of interest. GradientOptimizer is straightforward to use, requires minimum processing of the input files, and is supported under Windows, Linux, and OS X platforms. The software is open-source and can be downloaded under an Apache 2.0 license at https://github.com/statisticalbiotechnology/NonlinearGradientsUI.

  • 56. Moruz, Luminita
    et al.
    Käll, Lukas
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, Centres, SeRC - Swedish e-Science Research Centre.
    GradientOptimizer: An open-source graphical environment for calculating optimized gradients in reversed-phase liquid chromatography2014In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 14, no 12, p. 1464-1466Article in journal (Refereed)
    Abstract [en]

    We here present GradientOptimizer, an intuitive, lightweight graphical user interface to design nonlinear gradients for separation of peptides by reversed-phase liquid chromatography. The software allows to calculate three types of nonlinear gradients, each of them optimizing a certain retention time distribution of interest. GradientOptimizer is straightforward to use, requires minimum processing of the input files, and is supported under Windows, Linux, and OS X platforms. The software is open-source and can be downloaded under an Apache 2.0 license at https://github.com/statisticalbiotechnology/NonlinearGradientsUI.

  • 57.
    Moruz, Luminita
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Staes, An
    Foster, Joseph M.
    Hatzou, Maria
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Timmerman, Evy
    Martens, Lennart
    Kall, Lukas
    Chromatographic retention time prediction for posttranslationally modified peptides2012In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 12, no 8, p. 1151-1159Article in journal (Refereed)
    Abstract [en]

    Retention time prediction of peptides in liquid chromatography has proven to be a valuable tool for mass spectrometry-based proteomics, especially in designing more efficient procedures for state-of-the-art targeted workflows. Additionally, accurate retention time predictions can also be used to increase confidence in identifications in shotgun experiments. Despite these obvious benefits, the use of such methods has so far not been extended to (posttranslationally) modified peptides due to the absence of efficient predictors for such peptides. We here therefore describe a new retention time predictor for modified peptides, built on the foundations of our existing Elude algorithm. We evaluated our software by applying it on five types of commonly encountered modifications. Our results show that Elude now yields equally good prediction performances for modified and unmodified peptides, with correlation coefficients between predicted and observed retention times ranging from 0.93 to 0.98 for all the investigated datasets. Furthermore, we show that our predictor handles peptides carrying multiple modifications as well. This latest version of Elude is fully portable to new chromatographic conditions and can readily be applied to other types of posttranslational modifications. Elude is available under the permissive Apache2 open source License at or can be run via a web-interface at .

  • 58. Moruz, Luminita
    et al.
    Staes, An
    Foster, Joseph M.
    Hatzou, Maria
    Timmerman, Evy
    Martens, Lennart
    Käll, Lukas
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Chromatographic retention time prediction for posttranslationally modified peptides2012In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 12, no 8, p. 1151-1159Article in journal (Refereed)
    Abstract [en]

    Retention time prediction of peptides in liquid chromatography has proven to be a valuable tool for mass spectrometry-based proteomics, especially in designing more efficient procedures for state-of-the-art targeted workflows. Additionally, accurate retention time predictions can also be used to increase confidence in identifications in shotgun experiments. Despite these obvious benefits, the use of such methods has so far not been extended to (posttranslationally) modified peptides due to the absence of efficient predictors for such peptides. We here therefore describe a new retention time predictor for modified peptides, built on the foundations of our existing Elude algorithm. We evaluated our software by applying it on five types of commonly encountered modifications. Our results show that Elude now yields equally good prediction performances for modified and unmodified peptides, with correlation coefficients between predicted and observed retention times ranging from 0.93 to 0.98 for all the investigated datasets. Furthermore, we show that our predictor handles peptides carrying multiple modifications as well. This latest version of Elude is fully portable to new chromatographic conditions and can readily be applied to other types of posttranslational modifications. Elude is available under the permissive Apache2 open source License at or can be run via a web-interface at.

  • 59.
    Moulder, Robert
    et al.
    Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland.
    Filén, Jan-Jonas
    Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland; The National Graduate School in Informational and Structural Biology, Finland.
    Salmi, Jussi
    Department of Information Technology and Turku Centre for Computer Science, Turku, Finland.
    Katajamaa, Mikko
    Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland.
    Nevalainen, Olli S.
    Department of Information Technology and Turku Centre for Computer Science, Turku, Finland.
    Oresic, Matej
    VTT Biotechnology, Espoo, Finland.
    Aittokallio, Tero
    Department of Mathematics, University of Turku, Finland.
    Lahesmaa, Riitta
    Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland.
    Nyman, Tuula A.
    A comparative evaluation of software for the analysis of liquid chromatography-tandem mass spectrometry data from isotope coded affinity tag experiments2005In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 5, no 11, p. 2748-2760Article in journal (Refereed)
    Abstract [en]

    The options available for processing quantitative data from isotope coded affinity tag (ICAT) experiments have mostly been confined to software specific to the instrument of acquisition. However, recent developments with data format conversion have subsequently increased such processing opportunities. In the present study, data sets from ICAT experiments, analysed with liquid chromatography/tandem mass spectrometry (MS/MS), using an Applied Biosystems QSTAR Pulsar quadrupole-TOF mass spectrometer, were processed in triplicate using separate mass spectrometry software packages. The programs Pro ICAT, Spectrum Mill and SEQUEST with XPRESS were employed. Attention was paid towards the extent of common identification and agreement of quantitative results, with additional interest in the flexibility and productivity of these programs. The comparisons were made with data from the analysis of a specifically prepared test mixture, nine proteins at a range of relative concentration ratios from 0.1 to 10 (light to heavy labelled forms), as a known control, and data selected from an ICAT study involving the measurement of cytokine induced protein expression in human lymphoblasts, as an applied example. Dissimilarities were detected in peptide identification that reflected how the associated scoring parameters favoured information from the MS/MS data sets. Accordingly, there were differences in the numbers of peptides and protein identifications, although from these it was apparent that both confirmatory and complementary information was present. In the quantitative results from the three programs, no statistically significant differences were observed.

  • 60.
    Navakauskiene, R.
    et al.
    Department of Developmental Biology, Institute of Biochemistry, Vilnius, Lithuania, Department of Developmental Biology, Institute of Biochemistry, Mokslininku 12, LT-08662 Vilnius, Lithuania.
    Treigyte, G.
    Department of Developmental Biology, Institute of Biochemistry, Vilnius, Lithuania.
    Gineitis, A.
    Department of Developmental Biology, Institute of Biochemistry, Vilnius, Lithuania.
    Magnusson, Karl-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology .
    Identification of apoptotic tyrosine-phosphorylated proteins after etoposide or retinoic acid treatment of HL-60 cells2004In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 4, no 4, p. 1029-1041Article in journal (Refereed)
    Abstract [en]

    A main shortcoming of using HL-60 cells as a model of granulocyte-macrophage differentiation is that some cells in the differentiating population undergo apoptosis. To address this issue, we have identified which tyrosine-phosphorylated proteins are involved in apoptosis and differentiation, respectively. HL-60 cells were induced specifically to undergo apoptosis with 68 µM etoposide, and to undergo granulocytic differentiation with 1 µM retinoic acid (RA). The corresponding two-dimensional electrophoretic maps of tyrosine-phosphorylated proteins from treated cells were compared. In the 8 h etoposide-treated HL-60 cell population, 83% of the cells were apoptotic. In the 120 h RA-treated cells, 50% of the cells were apoptotic. Eighteen cytosolic and nuclear tyrosine-phosphorylated proteins were found in both the 8 h etoposide- and the 120 h RA-treated cells, but not in the proliferating HL-60 cell population. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry analyses suggested that some of the proteins may be involved in signal transduction pathways (NF?B, GTP-binding protein, protein disulfide isomerase, Cyclophilin A), others in cell transcriptional and translational control (hnRNP H, hnRNP L, Hsp60, Hp1, Hcc-1, 26S proteasome beta-subunit, ATP synthase beta-chain), and a third group in cell cytoskeleton organization and receptor cycling (profilin, caveolin-1). An understanding of signal transduction in apoptosis initiation by screening for tyrosine-phosphorylated proteins associated with apoptosis may provide new targets for the treatment of leukemia.

  • 61.
    Neiman, Maja
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Fredolini, Claudia
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Johansson, H.
    Lehtiö, Janne
    Karolinska Institutet, Solna, Sweden .
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Protein Technology.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Schwenk, Jochen M.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Selectivity analysis of single binder assays used in plasma protein profiling2013In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 13, no 23-24, p. 3406-3410Article in journal (Refereed)
    Abstract [en]

    The increasing availability of antibodies toward human proteins enables broad explorations of the proteomic landscape in cells, tissues, and body fluids. This includes assays with antibody suspension bead arrays that generate protein profiles of plasma samples by flow cytometer analysis. However, antibody selectivity is context dependent so it is necessary to corroborate on-target detection over off-target binding. To address this, we describe a concept to directly verify interactions from antibody-coupled beads by analysis of their eluates by Western blots and MS. We demonstrate selective antibody binding in complex samples with antibodies toward a set of chosen proteins with different abundance in plasma and serum, and illustrate the need to adjust sample and bead concentrations accordingly. The presented approach will serve as an important tool for resolving differential protein profiles from antibody arrays within plasma biomarker discoveries.

  • 62.
    Nilsson, Per
    et al.
    RIKEN, Brain Sci Inst, Lab Proteolyt Neurosci, Saitama, Japan.;Karolinska Inst, Ctr Alzheimer Res, Dept Neurobiol Care Sci & Soc, Div Neurogeriatr, Huddinge, Sweden..
    Loganathan, Krishnapriya
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology. RIKEN, Brain Sci Inst, Lab Proteolyt Neurosci, Saitama, Japan.;Karolinska Inst, Ctr Alzheimer Res, Dept Neurobiol Care Sci & Soc, Div Neurogeriatr, Huddinge, Sweden..
    Sekiguchi, Misaki
    RIKEN, Brain Sci Inst, Lab Proteolyt Neurosci, Saitama, Japan..
    Winblad, Bengt
    Karolinska Inst, Ctr Alzheimer Res, Dept Neurobiol Care Sci & Soc, Div Neurogeriatr, Huddinge, Sweden..
    Iwata, Nobuhisa
    Nagasaki Univ, Grad Sch Biomed Sci, Dept Biotechnol, Nagasaki 852, Japan..
    Saido, Takaomi C.
    RIKEN, Brain Sci Inst, Lab Proteolyt Neurosci, Saitama, Japan..
    Tjernberg, Lars O.
    Karolinska Inst, Ctr Alzheimer Res, Dept Neurobiol Care Sci & Soc, Div Neurogeriatr, Huddinge, Sweden..
    Loss of neprilysin alters protein expression in the brain of Alzheimer's disease model mice2015In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 15, no 19, p. 3349-3355Article in journal (Refereed)
    Abstract [en]

    Alzheimer's disease (AD) is a neurodegenerative disease displaying extracellular plaques formed by the neurotoxic amyloid -peptide (A), and intracellular neurofibrillary tangles consisting of protein tau. However, how these pathologies relate to the massive neuronal death that occurs in AD brains remain elusive. Neprilysin is the major A-degrading enzyme and a lack thereof increases A levels in the brain twofold. To identify altered protein expression levels induced by increased A levels, we performed a proteomic analysis of the brain of the AD mouse model APPsw and compared it to that of APPsw mice lacking neprilysin. To this end we established an LC-MS/MS method to analyze brain homogenate, using an O-18-labeled internal standard to accurately quantify the protein levels. To distinguish between alterations in protein levels caused by increased A levels and those induced by neprilysin deficiency independently of A, the brain proteome of neprilysin deficient APPsw mice was also compared to that of neprilysin deficient mice. By this approach we identified approximately 600 proteins and the levels of 300 of these were quantified. Pathway analysis showed that many of the proteins with altered expression were involved in neurological disorders, and that tau, presenilin and APP were key regulators in the identified networks. The data have been deposited to the ProteomeXchange Consortium with identifiers PXD000968 and PXD001786 ( and (). Interestingly, the levels of several proteins, including some not previously reported to be linked to AD, were associated with increased A levels.

  • 63.
    Nilsson, Peter
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Larsson, Karin
    KTH, School of Biotechnology (BIO).
    Persson, Anja
    KTH, School of Biotechnology (BIO), Proteomics.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Wernérus, Henrik
    KTH, School of Biotechnology (BIO).
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Björling, Erik
    KTH, School of Biotechnology (BIO).
    Ottoson, Jenny
    KTH, School of Biotechnology (BIO), Proteomics.
    Ödling, Jenny
    KTH, School of Biotechnology (BIO).
    Sundberg, Mårten
    KTH, School of Biotechnology (BIO), Proteomics.
    Al-Khalili Szigyarto, Cristina
    KTH, School of Biotechnology (BIO), Proteomics.
    Paavilainen, Linda
    Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
    Andersson, Ann-Catrin
    Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
    Kampf, Caroline
    Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
    Wester, Kenneth
    Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
    Pontén, Fredrik
    Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
    Towards a human proteome atlas: High-throughput generation of mono-specific antibodies for tissue profiling2005In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 5, p. 4327-4337Article in journal (Refereed)
    Abstract [en]

    A great need exists for the systematic generation of specific antibodies to explore the human proteome. Here, we show that antibodies specific to human proteins can be generated in a high-throughput manner involving stringent affinity purification using recombinant protein epitope signature tags (PrESTs) as immunogens and affinity-ligands. The specificity of the generated affinity reagents, here called mono-specific antibodies (msAb), were validated with a novel protein microarray assay. The success rate for 464 antibodies generated towards human proteins was more than 90% as judged by the protein array assay. The antibodies were used for parallel profiling of patient biopsies using tissue microarrays generated from 48 human tissues. Comparative analysis with well-characterized monoclonal antibodies showed identical or similar specificity and expression patterns. The results suggest that a comprehensive atlas containing extensive protein expression and subcellular localization data of the human proteome can be generated in an efficient manner with mono-specific antibodies.

  • 64. Nilsson, Peter
    et al.
    Paavilainen, Linda
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Larsson, Karin
    Ödling, Jenny
    Sundberg, Mårten
    Department of Biotechnology, Royal Institute of Technology (KTH), Stockholm.
    Andersson, Ann-Catrin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Kampf, Caroline
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Persson, Anja
    Al-Khalili Szigyarto, Cristina
    Ottosson, Jenny
    Björling, Erik
    Hober, Sophia
    Wernérus, Henrik
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Ponten, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Uhlen, Mathias
    Towards a human proteome atlas: high-throughput generation of mono-specific antibodies for tissue profiling2005In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 5, no 17, p. 4327-4337Article in journal (Refereed)
    Abstract [en]

    A great need exists for the systematic generation of specific antibodies to explore the human proteome. Here, we show that antibodies specific to human proteins can be generated in a high-throughput manner involving stringent affinity purification using recombinant protein epitope signature tags (PrESTs) as immunogens and affinity-ligands. The specificity of the generated affinity reagents, here called mono-specific antibodies (msAb), were validated with a novel protein microarray assay. The success rate for 464 antibodies generated towards human proteins was more than 90% as judged by the protein array assay. The antibodies were used for parallel profiling of patient biopsies using tissue microarrays generated from 48 human tissues. Comparative analysis with well-characterized monoclonal antibodies showed identical or similar specificity and expression patterns. The results suggest that a comprehensive atlas containing extensive protein expression and subcellular localization data of the human proteome can be generated in an efficient manner with mono-specific antibodies.

  • 65. Nolte, Hendrik
    et al.
    Hoelper, Soraya
    Housley, Michael P.
    Islam, Shariful
    Piller, Tanja
    Konzer, Anne
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Stainier, Didier Y. R.
    Braun, Thomas
    Krueger, Marcus
    Dynamics of zebrafish fin regeneration using a pulsed SILAC approach2015In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 15, no 4, p. 739-751Article in journal (Refereed)
    Abstract [en]

    The zebrafish owns remarkable regenerative capacities allowing regeneration of several tissues, including the heart, liver, and brain. To identify protein dynamics during fin regeneration we used a pulsed SILAC approach that enabled us to detect the incorporation of C-13(6)-lysine (Lys6) into newly synthesized proteins. Samples were taken at four different time points from noninjured and regrowing fins and incorporation rates were monitored using a combination of single-shot 4-h gradients and high-resolution tandem MS. We identified more than 5000 labeled proteins during the first 3 weeks of fin regeneration and were able to monitor proteins that are responsible for initializing and restoring the shape of these appendages. The comparison of Lys6 incorporation rates between noninjured and regrowing fins enabled us to identify proteins that are directly involved in regeneration. For example, we observed increased incorporation rates of two actinodin family members at the actinotrichia, which is a hairlike fiber structure at the tip of regrowing fins. Moreover, we used quantitative real-time RNA measurements of several candidate genes, including osteoglycin, si:ch211-288h17.3, and prostaglandin reductase 1 to correlate the mRNA expression to Lys6 incorporation data. This novel pulsed SILAC methodology in fish can be used as a versatile tool to monitor newly synthesized proteins and will help to characterize protein dynamics during regenerative processes in zebrafish beyond fin regeneration.

  • 66.
    Nyblom, Hanna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Thorn, Kristofer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Ahmed, Meftun
    Bergsten, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Mitochondrial protein patterns correlating with impaired insulin secretion from INS-1E cells exposed to elevated glucose concentrations2006In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 6, no 19, p. 5193-5198Article in journal (Refereed)
    Abstract [en]

    Extended hyperglycaemia leads to impaired glucose-stimulated insulin secretion (GSIS) and eventually P-cell apoptosis in individuals with type 2 diabetes mellitus. In an attempt to dissect mechanisms behind the detrimental effects of glucose, we focused on measuring changes in expression patterns of mitochondrial proteins. Impaired GSIS was observed from INS-1E cells cultured for 5 days at 20 or 27 mM glucose compared to cells cultured at 5.5 or 11 mM glucose. After culture, mitochondria were isolated from the INS-1E cells by differential centrifugation. Proteins of the mitochondrial fraction were bound to a strong anionic surface (SAX2) protein array and mass spectra generated by SELDI-TOF-MS. Analysis of the spectra revealed proteins with expression levels that correlated with the glucose concentration of the culture medium. Indeed, such differentially expressed proteins created patterns of protein changes, which correlated with impairment of GSIS. In conclusion, the study reveals the first glucose-induced differentially expressed patterns of P-cell mitochondrial proteins obtained by SELDI-TOF-MS.

  • 67. Orchard, S.
    et al.
    Heck, A.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Ping, P.
    Publication Committee Meeting HUPO 5th Annual World Congress Long Beach, CA, USA 30 October 20062007In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 7, no 7, p. 1009-1011Article in journal (Refereed)
    Abstract [en]

    This meeting brought together delegates from industry, academia and the publishing houses to facilitate discussions on the level of support from the journals for the use of standardised data formats and their interest in the creation of a network of proteomics repositories collaborating on a coordinated data curation effort. Discussions centred on how best to structure interactions between journals, databases and researchers to improve accessibility to data, and facilitate comparisons between datasets.

  • 68.
    Ortsäter, Henrik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Sundsten, Tea
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Lin, Jian-Man
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Bergsten, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Evaluation of the SELDI-TOF MS technique for protein profiling of pancreatic islets exposed to glucose and oleate2007In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 7, no 17, p. 3105-3115Article in journal (Refereed)
    Abstract [en]

    The aim of the study was to evaluate the SELDI-TOF MS technique for pancreatic islet research. Mouse islets were cultured at low or high glucose levels in the absence or presence of oleate and characterized by measuring insulin secretion and oxygen tension. Subsequently, the islets were protein profiled. Up to 200 different peaks could be detected in a single experiment with the majority of peaks corresponding to proteins with masses below 30 kDa. By combining different protein arrays, the number of detected peaks could be increased further. The optimal binding of islet proteins was achieved using the anionic exchange array and phosphate buffer (pH 6) when the binding of insulin was low, which allowed other less abundant proteins to be captured. When islets from different culture conditions were profiled and analyzed, in total 25 proteins were found to be oleate/glucose-regulated. An oleate-regulated protein was chosen for identification work, which was conducted by passive elution from SDS-PAGE gels and subsequent in-gel trypsin digestion and MALDI-TOF MS. The protein was identified as peptidyl-prolyl isomerase B (PPI-B). In conclusion, the study demonstrates that SELDI-technique can be used not only to obtain islet protein patterns but is also helpful in the subsequent identification of differentially expressed proteins.

  • 69.
    Pang, Zhili
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience. China Agricultural University, China.
    Chen, Lei
    Miao, Jianqiang
    Wang, Zhiwen
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience. University of Adelaide, Australia.
    Liu, Xili
    Proteomic profile of the plant-pathogenic oomycete Phytophthora capsici in response to the fungicide pyrimorph2015In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 15, no 17, p. 2972-2982Article in journal (Refereed)
    Abstract [en]

    Pyrimorph is a novel fungicide from the carboxylic acid amide (CAA) family used to control plant-pathogenic oomycetes such as Phytophthora capsici. The proteomic response of P. capsici to pyrimorph was investigated using the iTRAQ technology to determine the target site of the fungicide and potential biomarker candidates of drug efficacy. A total of 1336 unique proteins were identified from the mycelium of wild-type P. capsici isolate (Hd3) and two pyrimorphresistantmutants (R3-1 and R3-2) grown in the presence or absence of pyrimorph. Comparative analysis revealed that the three P. capsici isolates Hd3, R3-1, and R3-2 produced 163, 77, and 13 unique proteins, respectively, which exhibited altered levels of abundance in response to the pyrimorph treatment. Further investigations, using Cluster of Orthologous Groups of Proteins (COG) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified 35 proteins related to the mode of action of pyrimorph against P. capsici and 62 proteins involved in the stress response of P. capsici to pyrimorph. Many of the proteins with altered expression were associated with glucose and energy metabolism. Biochemical analysis using D-[U-C-14] glucose verified the proteomics data, suggesting that the major mode of action of pyrimorph in P. capsici is the inhibition of cell wall biosynthesis. These results also illustrate that proteomics approaches are useful tools for determining the pathways targeted by novel fungicides as well as for evaluating the tolerance of plant pathogens to environmental challenges, such as the presence of fungicides.

  • 70.
    Piltti, Juha
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland.
    Varjosalo, Markku
    Institute of Biotechnology, University of Helsinki, Helsinki.
    Qu, Chengjuan
    Department of Applied Physics, University of Eastern Finland, Kuopio, Finland.
    Häyrinen, Jukka
    Institute of Biomedicine, University of Eastern Finland, Kuopio, Finland.
    Lammi, Mikko
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Rho-kinase inhibitor Y-27632 increases cellular proliferation and migration in human foreskin fibroblast cells2015In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 15, no 17, p. 2953-2965Article in journal (Refereed)
    Abstract [en]

    The idea of direct differentiation of somatic cells into other differentiated cell types has attracted a great interest recently. Rho-kinase inhibitor Y-27632 (ROCKi) is a potential drug molecule, which has been reported to support the gene expressions typical for the chondrocytes, thus restricting their phenotypic conversion to fibroblastic cells upon the cellular expansion. In this study, we have investigated the short-term biological responses of ROCKi to human primary foreskin fibroblasts. The fibroblast cells were exposed to 1 and 10 μM ROCKi treatments. A proteomics analysis revealed expression changes of 56 proteins, and a further protein pathway analysis suggested their association with the cell morphology, the organization, and the increased cellular movement and the proliferation. These functional responses were confirmed by a Cell-IQ time-lapse imaging analysis. Rho-kinase inhibitor treatment increased the cellular proliferation up to twofold during the first 12 h, and a wound model based migration assay showed 50% faster filling of the mechanically generated wound area. Additionally, significantly less vinculin-associated focal adhesions were present in the ROCKi-treated cells. Despite the marked changes in the cell behavior, ROCKi was not able to induce the expression of the chondrocyte-specific genes, such as procollagen α1 (II) and aggrecan.

  • 71.
    Potrykus, Joanna
    et al.
    Umeå University.
    Jonna, Venkateswara Rao
    Umeå University.
    Dopson, Mark
    Umeå University.
    Iron homeostasis and responses to iron limitation in extreme acidophiles from the Ferroplasma genus.2011In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 11, no 1, p. 52-63Article in journal (Refereed)
    Abstract [en]

    Extremely acidophilic archaea from the genus Ferroplasma inhabit iron-rich biomining environments and are important constituents of naturally occurring microbial consortia that catalyze the production of acid mine drainage. A combined bioinformatic, transcript profiling, and proteomic approach was used to elucidate iron homeostasis mechanisms in "F. acidarmanus" Fer1 and F. acidiphilum Y(T) . Bioinformatic analysis of the "F. acidarmanus" Fer1 genome sequence revealed genes encoding proteins hypothesized to be involved in iron-dependent gene regulation and siderophore biosynthesis; the Fhu and NRAMP cation acquisition systems; iron storage proteins; and the SUF machinery for the biogenesis of Fe-S clusters. A subset of homologous genes was identified on the F. acidiphilum Y(T) chromosome by direct PCR probing. In both strains, some of the genes appeared to be regulated in a ferrous/ferric iron-dependent manner, as indicated by RT-PCR. A detailed gel-based proteomics analysis of responses to iron depletion showed that a putative isochorismatase, presumably involved in siderophore biosynthesis, and the SufBCD system were upregulated under iron-limiting conditions. No evidence was obtained for iron sparing response during iron limitation. This study constitutes the first detailed investigation of iron homeostasis in extremely acidophilic archaea.

  • 72. Potrykus, Joanna
    et al.
    Jonna, Venkateswara Rao
    Dopson, Mark
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Iron homeostasis and responses to iron limitation in extreme acidophiles from the Ferroplasma genus2011In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 11, no 1, p. 52-63Article in journal (Refereed)
    Abstract [en]

    Extremely acidophilic archaea from the genus Ferroplasma inhabit iron-rich biomining environments and are important constituents of naturally occurring microbial consortia that catalyze the production of acid mine drainage. A combined bioinformatic, transcript profiling, and proteomic approach was used to elucidate iron homeostasis mechanisms in "F. acidarmanus" Fer1 and F. acidiphilum Y(T) . Bioinformatic analysis of the "F. acidarmanus" Fer1 genome sequence revealed genes encoding proteins hypothesized to be involved in iron-dependent gene regulation and siderophore biosynthesis; the Fhu and NRAMP cation acquisition systems; iron storage proteins; and the SUF machinery for the biogenesis of Fe-S clusters. A subset of homologous genes was identified on the F. acidiphilum Y(T) chromosome by direct PCR probing. In both strains, some of the genes appeared to be regulated in a ferrous/ferric iron-dependent manner, as indicated by RT-PCR. A detailed gel-based proteomics analysis of responses to iron depletion showed that a putative isochorismatase, presumably involved in siderophore biosynthesis, and the SufBCD system were upregulated under iron-limiting conditions. No evidence was obtained for iron sparing response during iron limitation. This study constitutes the first detailed investigation of iron homeostasis in extremely acidophilic archaea.

  • 73. Rhomberg, Thomas A.
    et al.
    Karlberg, Olof
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Mini, Thierry
    Zimny-Arndt, Ursula
    Wickenberg, Ulrika
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Röttgen, Marlene
    Jungblut, Peter
    Jenö, Paul
    Andersson, Siv
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Dehio, Christoph
    Proteomic analysis of the sarcosine-insoluble outer membrane fraction of the bacterial pathogen Bartonella henselae2004In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 4, no 10, p. 3021-3033Article in journal (Refereed)
    Abstract [en]

    Bartonella henselae is an emerging zoonotic pathogen causing a wide range of disease manifestations in humans. In this study, we report on the analysis of the sarcosine-insoluble outer membrane fraction of B. henselae ATCC 49882 Houston-1 by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1-D SDS-PAGE) and two-dimensional nonequilibrium pH gradient polyacrylamide gel electrophoresis (2-D NEPHGE). Protein species were identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and subsequent database query against the B. henselae genome sequence. Subcellular fractionation, application of the ionic detergent lauryl sarcosine, assessment of trypsin sensitivity, and heat modifiability of surface-exposed proteins represented valuable tools for the analysis of the outer membrane subproteome of B. henselae. 2-D NEPHGE was applied to display and catalogue a substantial number of proteins associated with the B. henselae sarcosine-insoluble outer membrane fraction, resulting in the establishment of a first 2-D reference map of this compartment. Thus, 53 distinct protein species associated with the outer membrane subproteome fraction were identified. This study provides novel insights into the membrane biology and the associated putative virulence factors of this pathogen of increasing medical importance.

  • 74.
    Sandh, Gustaf
    et al.
    Botaniska Institutionen Stockholms Universitet.
    Ran, Liang
    Botaniska Institutionen Stockholms Universitet.
    Xu, Linghua
    Botaniska Institutionen Stockholms Universitet.
    Sundqvist, Gustav
    KTH.
    Bulone, Vincent
    KTH.
    Bergman, Birgitta
    Botaniska Institutionen Stockholms Universitet.
    Comparative proteomic profiles of the marine cyanobacterium Trichodesmium erythraeum IMS101 under different nitrogen regimes.2011In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 11, no 3, p. 406-19Article in journal (Refereed)
    Abstract [en]

    Trichodesmium is a marine filamentous diazotrophic cyanobacterium and an important contributor of "new" nitrogen in the oligotrophic surface waters of the tropical and sub-tropical oceans. It is unique in that it exclusively fixes N(2) at daytime, although it belongs to the non-heterocystous filamentous segment of the cyanobacterial radiation. Here we present the first quantitative proteomic analysis of Trichodesmium erythraeum IMS101 when grown under different nitrogen regimes using 2-DE/MALDI-TOF-MS. Addition of combined nitrogen (NO3-) prevented development of the morphological characteristics of the N(2)-fixing cell type (diazocytes), inhibited expression of the nitrogenase enzyme subunits and consequently N(2) fixation activity. The diazotrophic regime (N(2) versus NO3- cultures) elicited the differential expression of more than 100 proteins, which represented 13.5% of the separated proteins. Besides proteins directly related to N(2) fixation, proteins involved in the synthesis of reducing equivalents and the generation of a micro-oxic environment were strongly up-regulated, as was in particular Dps, a protein related to iron acquisition and potentially other vital cellular processes. In contrast, proteins involved in the S-adenosylmethionine (SAM) cycle, synthesis of amino acids and production of carbon skeletons for storage and synthesis of amino acids were suppressed. The data are discussed in the context of Trichodesmium's unusual N(2)-fixing physiology.

  • 75.
    Sandh, Gustaf
    et al.
    Stockholm University, Faculty of Science, Department of Botany.
    Ran, Liang
    Stockholm University, Faculty of Science, Department of Botany.
    Xu, Linghua
    Stockholm University, Faculty of Science, Department of Botany.
    Sundqvist, Gustav
    Bulone, Vincent
    Bergman, Birgitta
    Stockholm University, Faculty of Science, Department of Botany.
    Comparative proteomic profiles of the marine cyanobacterium Trichodesmium erythraeum IMS101 under different nitrogen regimes2011In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 11, no 3, p. 406-419Article in journal (Refereed)
    Abstract [en]

    Trichodesmium is a marine filamentous diazotrophic cyanobacterium and an important contributor of new nitrogen in the oligotrophic surface waters of the tropical and subtropical oceans. It is unique in that it exclusively fixes N(2) at daytime, although it belongs to the non-heterocystous filamentous segment of the cyanobacterial radiation. Here we present the first quantitative proteomic analysis of Trichodesmium erythraeum IMS101 when grown under different nitrogen regimes using 2-DE/MALDI-TOF-MS. Addition of combined nitrogen (NO(3)(-)) prevented development of the morphological characteristics of the N(2)-fixing cell type (diazocytes), inhibited expression of the nitrogenase enzyme subunits and consequently N(2) fixation activity. The diazotrophic regime (N(2) versus NO3- cultures) elicited the differential expression of more than 100 proteins, which represented 13.5% of the separated proteins. Besides proteins directly related to N(2) fixation, proteins involved in the synthesis of reducing equivalents and the generation of a micro-oxic environment were strongly up-regulated, as was in particular Dps, a protein related to iron acquisition and potentially other vital cellular processes. In contrast, proteins involved in the S-adenosylmethionine (SAM) cycle, synthesis of amino acids and production of carbon skeletons for storage and synthesis of amino acids were suppressed. The data are discussed in the context of Trichodesmium's unusual N(2)-fixing physiology.

  • 76. Sandh, Gustaf
    et al.
    Ran, Liang
    Xu, Linghua
    Sundqvist, Gustav
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bergman, Birgitta
    Comparative proteomic profiles of the marine cyanobacterium Trichodesmium erythraeum IMS101 under different nitrogen regimes2011In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 11, no 3, p. 406-419Article in journal (Refereed)
    Abstract [en]

    Trichodesmium is a marine filamentous diazotrophic cyanobacterium and an important contributor of "new" nitrogen in the oligotrophic surface waters of the tropical and subtropical oceans. It is unique in that it exclusively fixes N-2 at daytime, although it belongs to the non-heterocystous filamentous segment of the cyanobacterial radiation. Here we present the first quantitative proteomic analysis of Trichodesmium erythraeum IMS101 when grown under different nitrogen regimes using 2-DE/MALDI-TOF-MS. Addition of combined nitrogen (NO3-) prevented development of the morphological characteristics of the N-2-fixing cell type (diazocytes), inhibited expression of the nitrogenase enzyme subunits and consequently N-2 fixation activity. The diazotrophic regime (N-2 versus NO3- cultures) elicited the differential expression of more than 100 proteins, which represented 13.5% of the separated proteins. Besides proteins directly related to N-2 fixation, proteins involved in the synthesis of reducing equivalents and the generation of a micro-oxic environment were strongly up-regulated, as was in particular Dps, a protein related to iron acquisition and potentially other vital cellular processes. In contrast, proteins involved in the S-adenosylmethionine (SAM) cycle, synthesis of amino acids and production of carbon skeletons for storage and synthesis of amino acids were suppressed. The data are discussed in the context of Trichodesmium's unusual N-2-fixing physiology.

  • 77. Schroetter, Andreas
    et al.
    Park, Young Mok
    Marcus, Katrin
    Martins-de-Souza, Daniel
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    El Magraoui, Fouzi
    Meyer, Helmut E.
    Grinberg, Lea T.
    Key players in neurodegenerative disorders in focus - New insights into the proteomic profile of Alzheimer's disease, schizophrenia, ALS, and multiple sclerosis24th HUPO BPP Workshop: September 29, 2015, Vancouver, Canada2016In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 16, no 7, p. 1047-1050Article in journal (Refereed)
    Abstract [en]

    The HUPO Brain Proteome Project (HUPO BPP) held its 24th workshop in Vancouver, Canada, September 29, 2015. The focus of the autumn workshop was on new insights into the proteomic profile of Alzheimer's disease, schizophrenia, ALS and multiple sclerosis.

  • 78. Schwarz, Daniel
    et al.
    Daley, Daniel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Beckhaus, Tobias
    Doetsch, Volker
    Bernhard, Frank
    Cell-free expression profiling of E. coli inner membrane proteins2010In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 10, no 9, p. 1762-1779Article in journal (Refereed)
    Abstract [en]

    The high versatility and open nature of cell-free expression systems offers unique options to modify expression environments. In particular for membrane proteins, the choice of co-translational versus post-translational solubilization approaches could significantly modulate expression efficiencies and even sample qualities. The production of a selection of 134 a-helical integral membrane proteins of the Escherichia cob inner membrane proteome focussing on larger transporters has therefore been evaluated by a set of individual cell-free expression reactions. The production profiles of the targets in different cell-free expression modes were analyzed independently by three screening strategies. Translational green fluorescent protein fusions were analyzed as monitor for the formation of proteomicelles after cell-free expression of membrane proteins in the presence of detergents. In addition, two different reaction configurations were implemented and performed either by robotic semi-throughput approaches or by individually designed strategies. The expression profiles were specified for the particular cell-free modes and overall, the production of 87% of the target list could be verified and approximately 50% could already be synthesized in preparative scales. The expression of several selected targets was up-scaled to milliliter volumes and milligram amounts of production. As an example, the flavocytochrome YedZ was purified and its sample quality was demonstrated.

  • 79.
    Schwenk, Jochen M.
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Igel, Ulrika
    KTH, School of Biotechnology (BIO), Proteomics.
    Kato, Bernet S.
    Nicholson, George
    Karpe, Fredrik
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics.
    Comparative protein profiling of serum and plasma using an antibody suspension bead array approach2010In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 10, no 3, p. 532-540Article in journal (Refereed)
    Abstract [en]

    In the pursuit towards a systematic analysis of human diseases, array-based approaches within antibody proteomics offer high-throughput strategies to discover protein biomarkers in serum and plasma. To investigate the influence of sample preparation on such discovery attempts, we report on a systematic effort to compare serum and plasma protein profiles determined with an antibody suspension bead array. The intensity levels were used to define protein profiles and no significant differences between serum and plasma were observed for 79% of the 174 antibodies (targeting 156 proteins). By excluding 36 antibodies giving rise to differential intensity levels, cluster analysis revealed donor-specific rather than preparation-dependent grouping. With a cohort from a clinically relevant medical condition, the metabolic syndrome, the influence of the sample type on a multiplexed biomarker discovery approach was further investigated. Independent comparisons of protein profiles in serum and plasma revealed an antibody targeting ADAMTSL-4, a protein that would qualify to be studied further in association with the condition. In general, the preparation type had an impact on the results of the applied antibody suspension bead array, and while the technical variability was equal, plasma offered a greater biological variability and allowed to give rise to more discoveries than serum.

  • 80.
    Sialana, Fernando J.
    et al.
    Univ Vienna, Dept Pharmaceut Chem, Vienna, Austria.;Austrian Acad Sci, CeMM Res Ctr Mol Med, Vienna, Austria..
    Gulyassy, Peter
    Eotvos Lorand Univ, Inst Biol, Lab Prote, Budapest, Hungary..
    Majek, Peter
    Austrian Acad Sci, CeMM Res Ctr Mol Med, Vienna, Austria..
    Sjöstedt, Evelina
    Uppsala University, Science for Life Laboratory, SciLifeLab. KTH Royal Inst Technol, Sch Biotechnol, Sci Life Lab, Stockholm, Sweden.
    Kis, Viktor
    Eotvos Lorand Univ, Dept Anat Cell & Dev Biol, Budapest, Hungary..
    Muller, Andre C.
    Austrian Acad Sci, CeMM Res Ctr Mol Med, Vienna, Austria.;ThermoFisher Sci, Vienna, Austria..
    Rudashevskaya, Elena L.
    Med Univ Vienna, Inst Med Chem, Vienna, Austria.;ISAS, Leibniz Inst Analyt Wissensch, Dortmund, Germany..
    Mulder, Jan
    Uppsala University, Science for Life Laboratory, SciLifeLab.
    Bennett, Keiryn L.
    Austrian Acad Sci, CeMM Res Ctr Mol Med, Vienna, Austria..
    Lubec, Gert
    Univ Vienna, Dept Pharmaceut Chem, Vienna, Austria..
    Mass spectrometric analysis of synaptosomal membrane preparations for the determination of brain receptors, transporters and channels2016In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 16, no 22, p. 2911-2920Article in journal (Refereed)
    Abstract [en]

    The molecular composition of synaptic signal transduction machineries shapes synaptic neurotransmission. The repertoire of receptors, transporters and channels (RTCs) comprises major signaling events in the brain. RTCs are conventionally studied by candidate immunohistochemistry and biochemistry, which are low throughput with resolution greatly affected by available immunoreagents and membrane interference. Therefore, a comprehensive resource of synaptic brain RTCs is still lacking. In particular, studies on the detergent-soluble synaptosomal fraction, known to contain transporters and channels, are limited. We, therefore, performed sub-synaptosomal fractionation of rat cerebral cortex, followed by trypsin/chymotrypsin sequential digestion of a detergent-soluble synaptosomal fraction and a postsynaptic density preparation, stable-isotope tryptic peptide labeling and liquid chromatography mass spectrometry. Based on the current study, a total of 4784 synaptic proteins were submitted to the ProteomExchange database (PXD001948), including 274 receptors, 394 transporters/channels and 1377 transmembrane proteins. Function-based classification assigned 1781 proteins as probable drug targets with 834 directly linked to brain disorders. The analytical approach identified 499 RTCs that are not listed in the largest, curated database for synaptosomal proteins (SynProt). This is a threefold RTC increase over all other data collected to date. Taken together, we present a protein discovery resource that can serve as a benchmark for future molecular interrogation of synaptic connectivity.

  • 81.
    Sialana, Fernando J.
    et al.
    Univ Vienna, Dept Pharmaceut Chem, Vienna, Austria.;Austrian Acad Sci, CeMM Res Ctr Mol Med, Vienna, Austria..
    Gulyassy, Peter
    Eotvos Lorand Univ, Inst Biol, Lab Prote, Budapest, Hungary..
    Majek, Peter
    Austrian Acad Sci, CeMM Res Ctr Mol Med, Vienna, Austria..
    Sjöstedt, Evelina
    KTH, Centres, Science for Life Laboratory, SciLifeLab. Uppsala Univ, Dept Immunol Genet & Pathol, Sci Life Lab, Uppsala, Sweden..
    Kis, Viktor
    Eotvos Lorand Univ, Dept Anat Cell & Dev Biol, Budapest, Hungary..
    Muller, Andre C.
    Austrian Acad Sci, CeMM Res Ctr Mol Med, Vienna, Austria.;ThermoFisher Sci, Vienna, Austria..
    Rudashevskaya, Elena L.
    Med Univ Vienna, Inst Med Chem, Vienna, Austria.;ISAS, Leibniz Inst Analyt Wissensch, Dortmund, Germany..
    Mulder, Jan
    Uppsala Univ, Dept Neurosci, Sci Life Lab, Uppsala, Sweden..
    Bennett, Keiryn L.
    Austrian Acad Sci, CeMM Res Ctr Mol Med, Vienna, Austria..
    Lubec, Gert
    Univ Vienna, Dept Pharmaceut Chem, Vienna, Austria..
    Mass spectrometric analysis of synaptosomal membrane preparations for the determination of brain receptors, transporters and channels2016In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 16, no 22, p. 2911-2920Article in journal (Refereed)
    Abstract [en]

    The molecular composition of synaptic signal transduction machineries shapes synaptic neurotransmission. The repertoire of receptors, transporters and channels (RTCs) comprises major signaling events in the brain. RTCs are conventionally studied by candidate immunohistochemistry and biochemistry, which are low throughput with resolution greatly affected by available immunoreagents and membrane interference. Therefore, a comprehensive resource of synaptic brain RTCs is still lacking. In particular, studies on the detergent-soluble synaptosomal fraction, known to contain transporters and channels, are limited. We, therefore, performed sub-synaptosomal fractionation of rat cerebral cortex, followed by trypsin/chymotrypsin sequential digestion of a detergent-soluble synaptosomal fraction and a postsynaptic density preparation, stable-isotope tryptic peptide labeling and liquid chromatography mass spectrometry. Based on the current study, a total of 4784 synaptic proteins were submitted to the ProteomExchange database (PXD001948), including 274 receptors, 394 transporters/channels and 1377 transmembrane proteins. Function-based classification assigned 1781 proteins as probable drug targets with 834 directly linked to brain disorders. The analytical approach identified 499 RTCs that are not listed in the largest, curated database for synaptosomal proteins (SynProt). This is a threefold RTC increase over all other data collected to date. Taken together, we present a protein discovery resource that can serve as a benchmark for future molecular interrogation of synaptic connectivity.

  • 82.
    Sköld, Karl
    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.
    Norrman, Mathias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Sjögren, Benita
    Svenningsson, Per
    Andrén, Per E
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    The significance of biochemical and molecular sample integrity in brain proteomics and peptidomics: Stathmin (2-20) and peptides as sample quality indicators.2007In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 7, no 24, p. 4445-4456Article in journal (Refereed)
    Abstract [en]

    Comparisons of transcriptional and translational expression in normal and abnormal states are important to reach an understanding of pathogenesis and pathophysiology. Maintaining the biochemical, molecular, and structural sample integrity is essential for correct sample comparisons. We demonstrate that both proteins and neuropeptides, including their PTMs, are subjected to massive degradation in the brain already 1 min postmortem. Further, markers for determining the integrity and status of a biological sample were identified. The protein fragment stathmin 2-20 correlated well with the general level of postmortem degradation and may serve as a sample quality indicator for future work, both in animal and human postmortem brains. Finally, a novel method for preventing degradation of proteins and peptides in postmortem tissue is presented using rapid and uniform conductive heat transfer on tissue prior to the actual sample preparation procedures, which enables the relatively low-abundant neuropeptides to remain intact, minimizes degradation of proteins by proteolysis, and conserves the PTMs of the neuropeptides.

  • 83.
    Souchelnytskyi, Serhiy
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Bridging proteomics and systems biology: What are the roads to be traveled?2005In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 5, no 16, p. 4123-4137Article in journal (Refereed)
    Abstract [en]

    The comprehensive study of proteomes has become an important part of attempts to uncover the systemic properties of biological systems. Proteomics provides data of a quality which increasingly fulfills strict requirements of systems biology for quantitative and qualitative information. Notably, proteomics can generate rich datasets that describe dynamic changes of proteomes. On the other hand, large-scale modeling requires the development of mathematic tools that are adequate for the processing of largely uncertain biological data. In this review, recent developments that pave the way for the integration of proteomics into systems biology are discussed. These developments include the standardization of data acquisition and presentation, the increased comprehensiveness of proteomics studies in description of functional status, localization and dynamics of proteins, and advanced modeling approaches.

  • 84.
    Souchelnytskyi, Serhiy
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Lomnytska, Marta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Dubrovska, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Volodko, N.
    Proteomics Success Story. Towards Early Detection of Breast and Ovarian Cancer: Plasma Proteomics as a Tool to Find Novel Markers2006In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 6, no S2, p. 65-68Article in journal (Refereed)
  • 85.
    Srivastava, Renu
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Pisareva, Tatiana
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Norling, Birgitta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Proteomic studies of the thylakoid membrane of Synechocystis sp. PCC 68032005In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 5, no 18, p. 4905-4916Article in journal (Refereed)
    Abstract [en]

    Purified thylakoid membranes from the cyanobacterium Synechocystis sp. PCC 6803 were used for the first time in proteomic studies. The membranes were prepared by a combination of sucrose density centrifugation and aqueous polymer two-phase partitioning. In total, 76 different proteins were identified from 2- and 1-D gels by MALDI-TOF MS analysis. Twelve of the identified proteins have a predicted Sec/Tat signal peptide. Fourteen of the proteins were known, or predicted to be, integral membrane proteins. Among the proteins identified were subunits of the well-characterized thylakoid membrane constituents Photosystem I and II, ATP synthase, cytochrome b6f-complex, NADH dehydrogenase, and phycobilisome complex. In addition, novel thylakoid membrane proteins, both integral and peripheral were found, including enzymes involved in protein folding and pigment biosynthesis. The latter were the chlorophyll biosynthesis enzymes, light-dependent protochlorophyllide reductase and geranylgeranyl reductase as well as phytoene desaturase involved in carotenoid biosynthesis and a water-soluble carotenoid-binding protein. Interestingly, in view of the protein sorting mechanism in cyanobacteria, one of the two signal peptidases type I of Synechocystis was found in the thylakoid membrane, whereas the second one has been identified previously in the plasma membrane. Sixteen proteins are hypothetical proteins with unknown function.

  • 86.
    Strömberg, Sara
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Gry Björklund, Marcus
    Asplund, Caroline
    Sköllermo, Anna
    Persson, Anja
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Kampf, Caroline
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Nilsson, Peter
    Andersson, Ann-Catrin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Uhlen, Mathias
    Kononen, Juha
    Pontén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Asplund, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    A high-throughput strategy for protein profiling in cell microarrays using automated image analysis2007In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 7, no 13, p. 2142-2150Article in journal (Refereed)
    Abstract [en]

    Advances in antibody production render a growing supply of affinity reagents for immunohistochemistry (IHC), and tissue microarray (TMA) technologies facilitate simultaneous analysis of protein expression in a multitude of tissues. However, collecting validated IHC data remains a bottleneck problem, as the standard method is manual microscopical analysis. Here we present a high-throughput strategy combining IHC on a recently developed cell microarray with a novel, automated image-analysis application (TMAx). The software was evaluated on 200 digital images of IHC-stained cell spots, by comparing TMAx annotation with manual annotation performed by seven human experts. A high concordance between automated and manual annotation of staining intensity and fraction of IHC-positive cells was found. In a limited study, we also investigated the possibility to assess the correlation between mRNA and protein levels, by using TMAx output results for relative protein quantification and quantitative real-time PCR for the quantification of corresponding transcript levels. In conclusion, automated analysis of immunohistochemically stained in vitro-cultured cells in a microarray format can be used for high-throughput protein profiling, and extraction of RNA from the same cell lines provides a basis for comparing transcription and protein expression on a global scale.

  • 87.
    Strömberg, Sara
    et al.
    Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University.
    Gry Björklund, Marcus
    KTH, School of Biotechnology (BIO), Proteomics.
    Asplund, Caroline
    KTH, School of Biotechnology (BIO), Proteomics.
    Sköllermo, Anna
    KTH, School of Biotechnology (BIO), Proteomics.
    Persson, Anja
    KTH, School of Biotechnology (BIO), Proteomics.
    Wester, Kenneth
    Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University.
    Kampf, Caroline
    Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics.
    Andersson, Ann-Catrin
    Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Kononen, Juha
    Beecher Instruments, Sun Prairie, WI, United States.
    Pontén, Fredrik
    Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University.
    Asplund, Anna
    Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University.
    A high-throughput strategy for protein profiling in cell microarrays using automated image analysis2007In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 7, no 13, p. 2142-2150Article in journal (Refereed)
    Abstract [en]

    Advances in antibody production render a growing supply of affinity reagents for immunohistochemistry (IHC), and tissue microarray (TMA) technologies facilitate simultaneous analysis of protein expression in a multitude of tissues. However, collecting validated IHC data remains a bottleneck problem, as the standard method is manual microscopical analysis. Here we present a high-throughput strategy combining IHC on a recently developed cell microarray with a novel, automated image-analysis application (TMAx). The software was evaluated on 200 digital images of IHC-stained cell spots, by comparing TMAx annotation with manual annotation performed by seven human experts. A high concordance between automated and manual annotation of staining intensity and fraction of IHC-positive cells was found. in a limited study, we also investigated the possibility to assess the correlation between mRNA and protein levels, by using TMAx output results for relative protein quantification and quantitative real-time PCR for the quantification of corresponding transcript levels. In conclusion, automated analysis of immunohistochemically stained in vitro-cultured cells in a microarray format can be used for high-throughput protein profiling, and extraction of RNA from the same cell lines provides a basis for comparing transcription and protein expression on a global scale.

  • 88.
    Strömbäck, Lena
    et al.
    Linköping University, Department of Computer and Information Science, Database and information techniques. Linköping University, The Institute of Technology.
    Hall, David
    Linköping University, Department of Computer and Information Science, Database and information techniques. Linköping University, The Institute of Technology.
    Lambrix, Patrick
    Linköping University, Department of Computer and Information Science, Database and information techniques. Linköping University, The Institute of Technology.
    A review of standards for data exchange within systems biology2007In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 7, no 6, p. 857-867Article in journal (Refereed)
    Abstract [en]

    The rapid increase in experimental data within systems biology has increased the need for exchange of data to allow analysis and comparison of larger datasets. This has resulted in a need for standardized formats for representation of such results and currently many formats for representation of data have been developed or are under development. In this paper, we give an overview of the current state of available standards and ontologies within systems biology. We focus on XML-based standards for exchange of data and give a thorough description of similarities and differences of currently available formats. For each of these, we discuss how the important concepts such as substances, interactions, and experimental data can be represented. In particular, we note that the purpose of a standard is often visible in the structures it provides for the representation of data. A clear purpose is also crucial for the success of a standard. Moreover, we note that the development of representation formats is parallel to the development of ontologies and the recent trend is that representation formats make more and more use of available ontologies.

  • 89.
    The, Matthew
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Tasnim, Ayesha
    KTH, School of Biotechnology (BIO), Gene Technology.
    Käll, Lukas
    KTH, School of Biotechnology (BIO), Gene Technology.
    How to talk about protein-level false discovery rates in shotgun proteomics2016In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 16, no 18, p. 2461-2469Article in journal (Refereed)
    Abstract [en]

    A frequently sought output from a shotgun proteomics experiment is a list of proteins that we believe to have been present in the analyzed sample before proteolytic digestion. The standard technique to control for errors in such lists is to enforce a preset threshold for the false discovery rate (FDR). Many consider protein-level FDRs a difficult and vague concept, as the measurement entities, spectra, are manifestations of peptides and not proteins. Here, we argue that this confusion is unnecessary and provide a framework on how to think about protein-level FDRs, starting from its basic principle: the null hypothesis. Specifically, we point out that two competing null hypotheses are used concurrently in today's protein inference methods, which has gone unnoticed by many. Using simulations of a shotgun proteomics experiment, we show how confusing one null hypothesis for the other can lead to serious discrepancies in the FDR. Furthermore, we demonstrate how the same simulations can be used to verify FDR estimates of protein inference methods. In particular, we show that, for a simple protein inference method, decoy models can be used to accurately estimate protein-level FDRs for both competing null hypotheses.

  • 90.
    Tsirigos, Konstantinos D.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Hennerdal, Aron
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Käll, Lukas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Elofsson, Arne
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    A guideline to proteome-wide alpha-helical membrane protein topology predictions2013In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 12, no 14, p. 2282-2294Article in journal (Refereed)
    Abstract [en]

    For current state-of-the-art methods, the prediction of correct topology of membrane proteins has been reported to be above 80%. However, this performance has only been observed in small and possibly biased data sets obtained from protein structures or biochemical assays. Here, we test a number of topology predictors on an unseen set of proteins of known structure and also on four genome-scale data sets, including one recent large set of experimentally validated human membrane proteins with glycosylated sites. The set of glycosylated proteins is also used to examine the ability of prediction methods to separate membrane from nonmembrane proteins. The results show that methods utilizing multiple sequence alignments are overall superior to methods that do not. The best performance is obtained by TOPCONS, a consensus method that combines several of the other prediction methods. The best methods to distinguish membrane from nonmembrane proteins belong to the Phobius group of predictors. We further observe that the reported high accuracies in the smaller benchmark sets are not quite maintained in larger scale benchmarks. Instead, we estimate the performance of the best prediction methods for eukaryotic membrane proteins to be between 60% and 70%. The low agreement between predictions from different methods questions earlier estimates about the global properties of the membrane proteome. Finally, we suggest a pipeline to estimate these properties using a combination of the best predictors that could be applied in large-scale proteomics studies of membrane proteins.

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  • 91. Turkina, Maria V
    et al.
    Blanco-Rivero, Amaya
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Vainonen, Julia P
    Vener, Alexander V
    Villarejo, Arsenio
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Department of Biology, Universidad Autónoma de Madrid, Spain.
    CO2 limitation induces specific redox-dependent protein phosphorylation in Chlamydomonas reinhardtii2006In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 6, no 9, p. 2693-2704Article in journal (Refereed)
    Abstract [en]

    Acclimation of the green alga Chlamydomonas reinhardtii to limiting environmental CO2 induced specific protein phosphorylation at the surface of photosynthetic thylakoid membranes. Four phosphopeptides were identified and sequenced by nanospray quadrupole TOF MS from the cells acclimating to limiting CO2. One phosphopeptide originated from a protein that has not been annotated. We found that this unknown expressed protein (UEP) was encoded in the genome of C. reinhardtii. Three other phosphorylated peptides belonged to Lci5 protein encoded by the low-CO2-inducible gene 5 (lci5). The phosphorylation sites were mapped in the tandem repeats of Lci5 ensuring phosphorylation of four serine and three threonine residues in the protein. Immunoblotting with Lci5-specific antibodies revealed that Lci5 was localized in chloroplast and confined to the stromal side of the thylakoid membranes. Phosphorylation of Lci5 and UEP occurred strictly at limiting CO2; it required reduction of electron carriers in the thylakoid membrane, but was not induced by light. Both proteins were phosphorylated in the low-CO2-exposed algal mutant deficient in the light-activated protein kinase Stt7. Phosphorylation of previously unknown basic proteins UEP and Lci5 by specific redox-dependent protein kinase(s) in the photosynthetic membranes reveals the early response of green algae to limitation in the environmental inorganic carbon.

  • 92. Walz, Anke
    et al.
    Odenbreit, Stefan
    Stühler, Kai
    Wattenberg, Andreas
    Meyer, Helmut E
    Mahdavi, Jafar
    Borén, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ruhl, Stefan
    Identification of glycoprotein receptors within the human salivary proteome for the lectin-like BabA and SabA adhesins of Helicobacter pylori by fluorescence-based 2-D bacterial overlay2009In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 9, no 6, p. 1582-1592Article in journal (Refereed)
    Abstract [en]

    Because gastric infection by Helicobacter pylori takes place via the oral route, possible interactions of this bacterium with human salivary proteins could occur. By using modified 1- and 2-D bacterial overlay, binding of H. pylori adhesins BabA and SabA to the whole range of salivary proteins was explored. Bound salivary receptor molecules were identified by MALDI-MS and by comparison to previously established proteome maps of whole and glandular salivas. By use of adhesin-deficient mutants, binding of H. pylori to MUC7 and gp-340 could be linked to the SabA and BabA adhesins, respectively, whereas binding to MUC5B was associated with both adhesins. Binding of H. pylori to the proline-rich glycoprotein was newly detected and assigned to BabA adhesin whereas the SabA adhesin was found to mediate binding to newly detected receptor molecules, including carbonic anhydrase VI, secretory component, heavy chain of secretory IgA1, parotid secretory protein and zinc-alpha(2)-glycoprotein. Some of these salivary glycoproteins are known to act as scavenger molecules or are involved in innate immunity whereas others might come to modify the pathogenetic properties of this organism. In general, this 2-D bacterial overlay technique represents a useful supplement in adhesion studies of bacteria with complex protein mixtures.

  • 93. Wen, Bo
    et al.
    Du, Chaoqin
    Li, Guilin
    Ghali, Fawaz
    Jones, Andrew R.
    Käll, Lukas
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, Centres, SeRC - Swedish e-Science Research Centre.
    Xu, Shaohang
    Zhou, Ruo
    Ren, Zhe
    Feng, Qiang
    Xu, Xun
    Wang, Jun
    IPeak: An open source tool to combine results from multiple MS/MS search engines2015In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 15, no 17, p. 2916-2920Article in journal (Refereed)
    Abstract [en]

    Liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) is an important technique for detecting peptides in proteomics studies. Here, we present an open source software tool, termed IPeak, a peptide identification pipeline that is designed to combine the Percolator post-processing algorithm and multi-search strategy to enhance the sensitivity of peptide identifications without compromising accuracy. IPeak provides a graphical user interface (GUI) as well as a command-line interface, which is implemented in JAVA and can work on all three major operating system platforms: Windows, Linux/Unix and OS X. IPeak has been designed to work with the mzIdentML standard from the Proteomics Standards Initiative (PSI) as an input and output, and also been fully integrated into the associated mzidLibrary project, providing access to the overall pipeline, as well as modules for calling Percolator on individual search engine result files. The integration thus enables IPeak (and Percolator) to be used in conjunction with any software packages implementing the mzIdentML data standard. IPeak is freely available and can be downloaded under an Apache 2.0 license at https://code.google.com/p/mzidentml-lib/.

  • 94.
    Wu, Chenglin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Comparative Physiology.
    Söderhäll, Irene
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Comparative Physiology.
    Kim, Young-A
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Comparative Physiology.
    Liu, Haipeng
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Comparative Physiology.
    Söderhäll, Kenneth
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Comparative Physiology.
    Hemocyte lineage marker proteins in a crustacean, the freshwater crayfish, Pacifastacus leniusculus2008In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 8, no 20, p. 4226-4235Article in journal (Refereed)
    Abstract [en]

    To identify proteins associated with development of different hemocyte types in the freshwater crayfish Pacifastacus leniusculus, 2-DE followed by MS analysis was carried out with hematopoietic tissue (Hpt) cells, semigranular cells (SGC) and granular cells (GC). Within the hemocyte lineages one two-domain Kazal proteinase inhibitor (KPI) was found to be specific for SGC, while a superoxide dismutase (SOD) was specific for GC at protein as well as at mRNA level. The proliferation cell nuclear antigen (PCNA) was detected at the mRNA level in Hpt cells only. We also provide evidence that SGC and GC most likely differentiate to maturation as separate lineages. We found that after laminarin or lipopolysaccharide (LPS) injection into crayfish, the transcript levels of PCNA and SOD increased in the Hpt cells, whereas the KPI transcript never was present in Hpt regardless of any challenge. RNA interference of PCNA in the Hpt cells led to that most of the cells did not spread or attach to the tissue culture dish. These results suggest that PCNA, KPI and SOD can be used as markers for Hpt cells, SGC and GC, respectively, and in conjunction with these results, a model is proposed how the Hpt responds to a microbial challenge by proliferation and release of Hpt cells.

  • 95.
    Wu, Chenglin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Söderhäll, Kenneth
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Söderhäll, Irene
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Two novel ficolin-like proteins act as pattern recognition receptors for invading pathogens in the freshwater crayfish Pacifastacus leniusculus2011In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 11, no 11, p. 2249-2264Article in journal (Refereed)
    Abstract [en]

    Abstract

    To isolate pathogen-associated molecular patterns (PAMPs)-binding molecules, the bacterium, Staphylococcus aureus was used as an affinity matrix to find bacteria binding proteins in the plasma of the freshwater crayfish, Pacifastacus leniusculus. Two new bacteria binding ficolin-like proteins (FLPs) were identified by 2-DE and MS analysis. The FLPs have a fibrinogen-related domain (FReD) in their C-terminal and a repeat region in their N-terminal region with putative structural similarities to the collagen-like domain of vertebrate ficolins and mannose binding lectins (MBLs). Phylogenetic analysis shows that the newly isolated crayfish FLP1 and FLP2 cluster separately from other FReD containing proteins. A tissue distribution study showed that the mRNA expression of FLP occurred mainly in the hematopoietic tissue (Hpt) and in the hepatopancreas. Recombinant FLPs exhibited agglutination activity of Gram-negative bacteria Escherichia coli and Aeromonas hydrophila in the presence of Ca2+. FLPs were able to bind to A. hydrophila, E.coli and S.aureus as judged by bacteria adsorption. Moreover, the FLPs may help crayfish to clear Gram-negative bacteria, but not Gram-positive bacteria which had been injected into the hemolymph. When Gram-negative bacteria coated with FLPs were incubated with Hpt cells, a lower death rate of the cells was found compared to control treatment. Our results suggest that FLPs function as pattern recognition receptors in the immune response of crayfish.

  • 96.
    Wåhlander, Åsa
    et al.
    Department of Protein Technology, Lund University, Lund, Sweden.
    Arrigoni, Giorgio
    Department of Biological Chemistry, University of Padova, Padova, Italy.
    Snel, Marten
    Waters Corporation Facilities, Manchester, UK.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    James, Peter
    Department of Protein Technology, Lund University, Lund, Sweden.
    Parallel post-source decay for increasing protein identification confidence levels from 2-D gels2008In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 8, no 9, p. 1771-1779Article in journal (Refereed)
    Abstract [en]

    Peptide mass fingerprinting (PMF) has over the years become one of the most commonly used tools for high-throughput analysis and identification of proteins. This method is applicable when relatively simple samples have to be analysed and it is commonly used for analysing proteins previously separated by 2-DE. The most common type of instrument used for this approach is the MALDI-TOF that has proved to be particularly suitable for the PMF analysis because of its characteristics of speed, robustness, sensitivity and automation. We have used a MALDI-TOF equipped with a novel parallel PSD capability (MALDI micro MX), to perform the analysis of two sets of different biological samples isolated by 2-DE. By using a method that integrates the data obtained by PMF analysis with the PSD data obtained in the same experiment, we show that the new multiplexed PSD solution increases the protein identification rate compared to the normal PMF approach. We also investigated the use of a charge-directed fragmentation modification reagent to improve the identification rate and confidence levels.

  • 97.
    Yentrapalli, Ramesh
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. Helmholtz Zentrum München .
    Azimzadeh, Omid
    Barjaktarovic, Zarko
    Sarioglu, Hakan
    Wojcik, Andrzej
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Harms-Ringdahl, Mats
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Atkinson, Michael J.
    Haghdoost, Siamak
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Tapio, Soile
    Quantitative proteomic analysis reveals induction of premature senescence in human umbilical vein endothelial cells exposed to chronic low-dose rate gamma radiation2013In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 13, no 7, p. 1096-1107Article in journal (Refereed)
    Abstract [en]

    Chronic low-dose ionizing radiation induces cardiovascular disease in human populations but the mechanism is largely unknown. We suggested that chronic radiation exposure may induce endothelial cell senescence that is associated with vascular damage in vivo. We investigated whether chronic radiation exposure is causing a change in the onset of senescence in endothelial cells in vitro. Indeed, when exposed to continuous low-dose rate gamma radiation (4.1 mGy/h), primary human umbilical vein endothelial cells (HUVECs) initiated senescence much earlier than the nonirradiated control cells. We investigated the changes in the protein expression of HUVECs before and during the onset of radiation-induced senescence. Cellular proteins were quantified using isotope-coded protein label technology after 1, 3, and 6 weeks of radiation exposure. Several senescence-related biological pathways were influenced by radiation, including cytoskeletal organization, cellcell communication and adhesion, and inflammation. Immunoblot analysis showed an activation of the p53/p21 pathway corresponding to the progressing senescence. Our data suggest that chronic radiation-induced DNA damage and oxidative stress result in induction of p53/p21 pathway that inhibits the replicative potential of HUVECs and leads to premature senescence. This study contributes to the understanding of the increased risk of cardiovascular diseases seen in populations exposed to chronic low-dose irradiation.

  • 98.
    Yu, Hans
    et al.
    Univ Bodenkultur Wien, Interuniv Dept Agrobiotechnol IFA Tulln, Inst Biotechnol Anim Prod, Tulln, Austria.;Univ Vet Med Vienna, Dept Biomed Sci, Inst Anim Breeding & Genet, Vienna, Austria..
    Reiser, Judith
    Ludwig Maximilians Univ Munchen, Inst Mol Anim Breeding & Biotechnol, Munich, Germany..
    Besenfelder, Urban
    Univ Bodenkultur Wien, Interuniv Dept Agrobiotechnol IFA Tulln, Inst Biotechnol Anim Prod, Tulln, Austria.;Univ Vet Med Vienna, Reprod Ctr Wieselburg, Vienna, Austria..
    Razzazi-Fazeli, Ebrahim
    Univ Vet Med Vienna, VetCore Facil Res, Vienna, Austria..
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Brem, Gottfried
    Univ Bodenkultur Wien, Interuniv Dept Agrobiotechnol IFA Tulln, Inst Biotechnol Anim Prod, Tulln, Austria.;Univ Vet Med Vienna, Dept Biomed Sci, Inst Anim Breeding & Genet, Vienna, Austria..
    Artemenko, Konstantin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Mayrhofer, Corina
    Univ Bodenkultur Wien, Interuniv Dept Agrobiotechnol IFA Tulln, Inst Biotechnol Anim Prod, Tulln, Austria.;Univ Vet Med Vienna, Dept Biomed Sci, Inst Anim Breeding & Genet, Vienna, Austria..
    Exploring the oviductal fluid proteome by a lectin-based affinity approach2016In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 16, no 23, p. 2962-2966Article in journal (Refereed)
    Abstract [en]

    The analysis of glycoproteins in body fluids represents a central task in the study of vital processes. Herein, we assessed the combined use of Concanavalin A and Wheat Germ Agglutinin as ligands to fractionate and enrich glycoproteins from oviductal fluid (OF), which is a source of molecules involved in fertilization. First, the selectivity was corroborated by a gel-based approach using glycoprotein staining and enzymatic deglycosylation. Nanoliquid chromatography-tandem mass spectrometry (nLC-ESI-MS/MS) further allowed the reliable identification of 134 nonbound as well as 130 lectin-bound OF proteins. Enrichment analysis revealed that 77% of the annotated proteins in the lectin-bound fraction were known glycoproteins (p-value [FDR] = 1.45E-31). The low variance of the number of peptide spectrum matches for each protein within replicates indicated a consistent reproducibility of the whole workflow (median CV 17.3% for technical replicates and 20.7% for biological replicates). Taken together, this study highlights the applicability of a lectin-based workflow for the comprehensive analysis of OF proteins and gives for the first time an insight into the broad glycoprotein content of OF.

  • 99. Öhman, Tiina
    et al.
    Söderholm, Sandra
    Paidikondala, Maruthibabu
    Lietzén, Niina
    Matikainen, Sampsa
    Nyman, Tuula A
    Phosphoproteome characterization reveals that Sendai virus infection activates mTOR signaling in human epithelial cells.2015In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 15, no 12, p. 2087-97Article in journal (Refereed)
    Abstract [en]

    Sendai virus (SeV) is a common respiratory pathogen in mice, rats, and hamsters. Host cell recognition of SeV is mediated by pathogen recognition receptors, which recognize viral components and induce intracellular signal transduction pathways that activate the antiviral innate immune response. Viruses use host proteins to control the activities of signaling proteins and their downstream targets, and one of the most important host protein modifications regulated by viral infection is phosphorylation. In this study, we used phosphoproteomics combined with bioinformatics to get a global view of the signaling pathways activated during SeV infection in human lung epithelial cells. We identified altogether 1347 phosphoproteins, and our data shows that SeV infection induces major changes in protein phosphorylation affecting the phosphorylation of almost one thousand host proteins. Bioinformatics analysis showed that SeV infection activates known pathways including MAPK signaling, as well as signaling pathways previously not linked to SeV infection including Rho family of GTPases, HIPPO signaling, and mammalian target of rapamycin (mTOR)-signaling pathway. Further, we performed functional studies with mTOR inhibitors and siRNA approach, which revealed that mTOR signaling is needed for both the host IFN response as well as viral protein synthesis in SeV-infected human lung epithelial cells.

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