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  • 1. Agaton, C.
    et al.
    Galli, J.
    Guthenberg, I. H.
    Janzon, L.
    Hansson, M.
    Asplund, A.
    Brundell, E.
    Lindberg, S.
    Ruthberg, I.
    Wester, K.
    Wurtz, D.
    Hoog, C.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Ståhl, Stefan
    KTH, Superseded Departments, Biotechnology.
    Ponten, F.
    Uhlén, Mathias
    KTH, Superseded Departments, Biotechnology.
    Affinity proteomics for systematic protein profiling of chromosome 21 gene products in human tissues2003In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 2, no 6, p. 405-414Article in journal (Refereed)
    Abstract [en]

    Here we show that an affinity proteomics strategy using affinity-purified antibodies raised against recombinant human protein fragments can be used for chromosome-wide protein profiling. The approach is based on affinity reagents raised toward bioinformatics-designed protein epitope signature tags corresponding to unique regions of individual gene loci. The genes of human chromosome 21 identified by the genome efforts were investigated, and the success rates for de novo cloning, protein production, and antibody generation were 85, 76, and 56%, respectively. Using human tissue arrays, a systematic profiling of protein expression and subcellular localization was undertaken for the putative gene products. The results suggest that this affinity proteomics strategy can be used to produce a proteome atlas, describing distribution and expression of proteins in normal tissues as well as in common cancers and other forms of diseased tissues.

  • 2. Ahmad, Yasmeen
    et al.
    Boisvert, Francois-Michel
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lamond, Angus I.
    Systematic Analysis of Protein Pools, Isoforms, and Modifications Affecting Turnover and Subcellular Localization2012In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 11, no 3Article in journal (Refereed)
    Abstract [en]

    In higher eukaryotes many genes encode protein isoforms whose properties and biological roles are often poorly characterized. Here we describe systematic approaches for detection of either distinct isoforms, or separate pools of the same isoform, with differential biological properties. Using information from ion intensities we have estimated protein abundance levels and using rates of change in stable isotope labeling with amino acids in cell culture isotope ratios we measured turnover rates and subcellular distribution for the HeLa cell proteome. Protein isoforms were detected using three data analysis strategies that evaluate differences between stable isotope labeling with amino acids in cell culture isotope ratios for specific groups of peptides within the total set of peptides assigned to a protein. The candidate approach compares stable isotope labeling with amino acids in cell culture isotope ratios for predicted isoform- specific peptides, with ratio values for peptides shared by all the isoforms. The rule of thirds approach compares the mean isotope ratio values for all peptides in each of three equal segments along the linear length of the protein, assessing differences between segment values. The three in a row approach compares mean isotope ratio values for each sequential group of three adjacent peptides, assessing differences with the mean value for all peptides assigned to the protein. Protein isoforms were also detected and their properties evaluated by fractionating cell extracts on one- dimensional SDS- PAGE prior to trypsin digestion and MS analysis and independently evaluating isotope ratio values for the same peptides isolated from different gel slices. The effect of protein phosphorylation on turnover rates was analyzed by comparing mean turnover values calculated for all peptides assigned to a protein, either including, or excluding, values for cognate phosphopeptides. Collectively, these experimental and analytical approaches provide a framework for expanding the func- tional annotation of the genome.

  • 3.
    Al-Khalili Szigyarto, Cristina
    et al.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Persson, A.
    KTH.
    Berglund, L.
    KTH.
    Tourle, S.
    KTH. Royal Inst Technol, Stockholm, Sweden..
    Ekstrom, M.
    KTH.
    Lindskog, M.
    KTH.
    Uhlén, Mathias
    KTH, Superseded Departments (pre-2005), Biotechnology.
    High throughput approach for bioinformatic design and cloning of protein epitope sequence tags suitable for antibody generation2005In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 4, no 8, p. S60-S60Article in journal (Other academic)
  • 4.
    Asplund, C.
    et al.
    KTH.
    Uhlén, Mathias
    KTH, Superseded Departments (pre-2005), Biotechnology. Royal Inst Technol, Stockholm, Sweden..
    Lundeberg, Joakim
    KTH.
    Persson, A.
    KTH.
    Real-time RT-PCR of protein epitope signature tags2005In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 4, no 8, p. S60-S60Article in journal (Other academic)
  • 5.
    Ayoglu, Burcu
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Häggmark, Anna
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Khademi, M.
    Olsson, T.
    Uhlén, Mathias
    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.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Autoantibody profiling in multiple sclerosis using arrays of human protein fragments2013In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 12, no 9, p. 2657-2672Article in journal (Refereed)
    Abstract [en]

    Profiling the autoantibody repertoire with large antigen collections is emerging as a powerful tool for the identification of biomarkers for autoimmune diseases. Here, a systematic and undirected approach was taken to screen for profiles of IgG in human plasma from 90 individuals with multiple sclerosis related diagnoses. Reactivity pattern of 11,520 protein fragments (representing ̃38% of all human protein encoding genes) were generated on planar protein microarrays built within the Human Protein Atlas. For more than 2,000 antigens IgG reactivity was observed, among which 64% were found only in single individuals. We used reactivity distributions among multiple sclerosis subgroups to select 384 antigens, which were then reevaluated on planar microarrays, corroborated with suspension bead arrays in a larger cohort (n = 376) and confirmed for specificity in inhibition assays. Among the heterogeneous pattern within and across multiple sclerosis subtypes, differences in recognition frequencies were found for 51 antigens, which were enriched for proteins of transcriptional regulation. In conclusion, using protein fragments and complementary high-throughput protein array platforms facilitated an alternative route to discovery and verification of potentially disease-associated autoimmunity signatures, that are now proposed as additional antigens for large-scale validation studies across multiple sclerosis biobanks.

  • 6.
    Barbe, L.
    et al.
    KTH.
    Lundberg, E.
    KTH.
    Brismar, H.
    KTH.
    Uhlén, Mathias
    KTH.
    Andersson, H.
    KTH.
    High-throughput confocal subcellular mapping for antibody-based proteomics2006In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 5, no 10, p. S240-S240Article in journal (Other academic)
  • 7. Bjorling, E.
    et al.
    Oksvold, P.
    KTH.
    Forsberg, M.
    Lund, J.
    Ponten, F.
    Uhlén, Mathias
    KTH.
    Human protein atlas, version 22006In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 5, no 10, p. S328-S328Article in journal (Other academic)
  • 8.
    Bjorling, E.
    et al.
    KTH.
    Oksvold, P.
    KTH.
    Forsberg, M.
    KTH.
    Lund, J.
    KTH.
    Ponten, F.
    Uppsala Univ, Uppsala, Sweden..
    Uhlén, Mathias
    KTH, Superseded Departments (pre-2005), Biotechnology.
    The creation and usage of a human protein atlas database2005In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 4, no 8, p. S18-S18Article in journal (Other academic)
  • 9. Buus, S.
    et al.
    Rockberg, Johan
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Forsström, Björn
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Schafer-Nielsen, C.
    High-resolution mapping of linear antibody epitopes using ultrahigh-density peptide microarrays2012In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 11, no 12, p. 1790-1800Article in journal (Refereed)
    Abstract [en]

    Antibodies empower numerous important scientific, clinical, diagnostic, and industrial applications. Ideally, the epitope(s) targeted by an antibody should be identified and characterized, thereby establishing antibody reactivity, highlighting possible cross-reactivities, and perhaps even warning against unwanted (e.g. autoimmune) reactivities. Antibodies target proteins as either conformational or linear epitopes. The latter are typically probed with peptides, but the cost of peptide screening programs tends to prohibit comprehensive specificity analysis. To perform high-throughput, high-resolution mapping of linear antibody epitopes, we have used ultrahigh-density peptide microarrays generating several hundred thousand different peptides per array. Using exhaustive length and substitution analysis, we have successfully examined the specificity of a panel of polyclonal antibodies raised against linear epitopes of the human proteome and obtained very detailed descriptions of the involved specificities. The epitopes identified ranged from 4 to 12 amino acids in size. In general, the antibodies were of exquisite specificity, frequently disallowing even single conservative substitutions. In several cases, multiple distinct epitopes could be identified for the same target protein, suggesting an efficient approach to the generation of paired antibodies. Two alternative epitope mapping approaches identified similar, although not necessarily identical, epitopes. These results show that ultrahigh-density peptide microarrays can be used for linear epitope mapping. With an upper theoretical limit of 2,000,000 individual peptides per array, these peptide microarrays may even be used for a systematic validation of antibodies at the proteomic level.

  • 10.
    Campuzano, Lain
    et al.
    Waters Corp, MS Technol, Manchester, Lancs, England..
    Brumer, Harry
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Piens, Kathleen
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Sage, Ashley
    Waters Corp, MS Technol, Manchester, Lancs, England..
    Mckenna, Therese
    Waters Corp, MS Technol, Manchester, Lancs, England..
    Langridge, Jim
    Waters Corp, MS Technol, Manchester, Lancs, England..
    Accurate mass analysis of glycoprotein isoforms by electrospray ionisation, orthogonal acceleration time-of-flight mass spectrometry and maximum entropy2004In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 3, no 10, p. S130-S130Article in journal (Other academic)
  • 11.
    DaCosta, R. S.
    et al.
    KTH.
    Lundberg, E.
    KTH.
    Constantinou, P.
    KTH.
    Asplund, A.
    KTH.
    Wilson, B. C.
    KTH.
    Ponten, F.
    KTH.
    Uhlén, Mathias
    KTH.
    Andersson, H.
    KTH.
    A novel confocal fluorescence MACROscope for high-throughput quantitative imaging of protein expression in cellular microarrays for biomarker and drug-target discovery2006In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 5, no 10, p. S168-S168Article in journal (Other academic)
  • 12. Dengjel, Joern
    et al.
    Hoyer-Hansen, Maria
    Nielsen, Maria O.
    Eisenberg, Tobias
    Harder, Lea M.
    Schandorff, Soren
    Farkas, Thomas
    Kirkegaard, Thomas
    Becker, Andrea C.
    Schroeder, Sabrina
    Vanselow, Katja
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nielsen, Mogens M.
    Kristensen, Anders R.
    Akimov, Vyacheslav
    Bunkenborg, Jakob
    Madeo, Frank
    Jaattela, Marja
    Andersen, Jens S.
    Identification of Autophagosome-associated Proteins and Regulators by Quantitative Proteomic Analysis and Genetic Screens2012In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 11, no 3Article in journal (Refereed)
    Abstract [en]

    Autophagy is one of the major intracellular catabolic pathways, but little is known about the composition of autophagosomes. To study the associated proteins, we isolated autophagosomes from human breast cancer cells using two different biochemical methods and three stimulus types: amino acid deprivation or rapamycin or concanamycin A treatment. The autophagosome- associated proteins were dependent on stimulus, but a core set of proteins was stimulus- independent. Remarkably, proteasomal proteins were abundant among the stimulus- independent common autophagosome- associated proteins, and the activation of autophagy significantly decreased the cellular proteasome level and activity supporting interplay between the two degradation pathways. A screen of yeast strains defective in the orthologs of the human genes encoding for a common set of autophagosome- associated proteins revealed several regulators of autophagy, including subunits of the retromer complex. The combined spatiotemporal proteomic and genetic data sets presented here provide a basis for further characterization of autophagosome biogenesis and cargo selection.

  • 13.
    Edfors, Fredrik
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Boström, Tove
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Forsström, Björn
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Zeiler, Marlis
    Johansson, Henrik J.
    Karlinska Institute.
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Protein Technology.
    Lehtiö, Janne
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Mann, Matthias
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Immunoproteomics using polyclonal antibodies and stable isotope-labeled affinity-purified recombinant proteins2014In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 13, no 6, p. 1611-1624Article in journal (Refereed)
    Abstract [en]

    AThe combination of immuno-based methods and mass spectrometry detection has great potential in the field of quantitative proteomics. Here, we describe a new method (immuno-SILAC) for the absolute quantification of proteins in complex samples based on polyclonal antibodies and stable isotope-labeled recombinant protein fragments to allow affinity enrichment prior to mass spectrometry analysis and accurate quantification. We took advantage of the antibody resources publicly available from the Human Protein Atlas project covering more than 80% of all human protein-coding genes. Epitope mapping revealed that a majority of the polyclonal antibodies recognized multiple linear epitopes, and based on these results, a semi-automated method was developed for peptide enrichment using polyclonal antibodies immobilized on protein A-coated magnetic beads. A protocol based on the simultaneous multiplex capture of more than 40 protein targets showed that approximately half of the antibodies enriched at least one functional peptide detected in the subsequent mass spectrometry analysis. The approach was further developed to also generate quantitative data via the addition of heavy isotope-labeled recombinant protein fragment standards prior to trypsin digestion. Here, we show that we were able to use small amounts of antibodies (50 ng per target) in this manner for efficient multiplex analysis of quantitative levels of proteins in a human HeLa cell lysate. The results suggest that polyclonal antibodies generated via immunization of recombinant protein fragments could be used for the enrichment of target peptides to allow for rapid mass spectrometry analysis taking advantage of a substantial reduction in sample complexity. The possibility of building up a proteome-wide resource for immuno-SILAC assays based on publicly available antibody resources is discussed.

  • 14. Ek, S.
    et al.
    Andreasson, U.
    Hober, Sophia
    KTH, School of Biotechnology (BIO).
    Kampf, Caroline
    KTH, School of Biotechnology (BIO).
    Ponten, Fredrik K.
    KTH, School of Biotechnology (BIO).
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO).
    Merz, H.
    Borrebaeck, C. A. K.
    From gene expression analysis to tissue microarrays - A rational approach to identify therapeutic and diagnostic targets in lymphoid malignancies2006In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 5, no 6, p. 1072-1081Article in journal (Refereed)
    Abstract [en]

    Mantle cell lymphoma (MCL) is an aggressive lymphoid malignancy for which better treatment strategies are needed. To identify potential diagnostic and therapeutic targets, a signature consisting of MCL-associated genes was selected based on a comprehensive gene expression analysis of malignant and normal B cells. The corresponding protein epitope signature tags were identified and used to raise monospecific, polyclonal antibodies, which were subsequently analyzed on paraffin-embedded sections of malignant and normal tissue. In this study, we demonstrate that the initial selection strategy of MCL-associated genes successfully allows identification of protein antigens either uniquely expressed or overexpressed in MCL compared with normal lymphoid tissues. We propose that genome-based, affinity proteomics, using protein epitope signature tag-induced antibodies, is an efficient way to rapidly identify a number of disease-associated protein candidates of both previously known and unknown identities.

  • 15.
    Eriksson, C.
    et al.
    KTH.
    Agaton, C.
    KTH.
    Kange, R.
    KTH.
    Nilsson, P.
    KTH.
    Uhlén, Mathias
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Gustafsson, M.
    KTH.
    Hober, Sophia
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Microfluidic analysis of antibodies in a compact disc format2005In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 4, no 8, p. S47-S47Article in journal (Other academic)
  • 16.
    Fagerberg, Linn
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hallström, Björn M.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Oksvold, Per
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Kampf, C.
    Djureinovic, D.
    Odeberg, Jacob
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Habuka, Masato
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Tahmasebpoor, S.
    Danielsson, A.
    Edlund, K.
    Asplund, A.
    Sjöstedt, E.
    Lundberg, E.
    Szigyarto, Cristina Al-Khalili
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Skogs, Marie
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ottosson Takanen, J.
    Berling, H.
    Tegel, Hanna
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Mulder, J.
    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.
    Lindskog, C.
    Danielsson, Frida
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Mardinoglu, A.
    Sivertsson, Åsa
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Von Feilitzen, Kalle
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Forsberg, Mattias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Zwahlen, Martin
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Olsson, I.
    Navani, S.
    Huss, Mikael
    Nielsen, Jens
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Pontén, F.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics2014In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 13, no 2, p. 397-406Article in journal (Refereed)
    Abstract [en]

    Global classification of the human proteins with regards to spatial expression patterns across organs and tissues is important for studies of human biology and disease. Here, we used a quantitative transcriptomics analysis (RNA-Seq) to classify the tissue-specific expression of genes across a representative set of all major human organs and tissues and combined this analysis with antibody- based profiling of the same tissues. To present the data, we launch a new version of the Human Protein Atlas that integrates RNA and protein expression data corresponding to 80% of the human protein-coding genes with access to the primary data for both the RNA and the protein analysis on an individual gene level. We present a classification of all human protein-coding genes with regards to tissue-specificity and spatial expression pattern. The integrative human expression map can be used as a starting point to explore the molecular constituents of the human body.

  • 17.
    Falk, Ronny
    et al.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Agaton, Charlotta
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Guthenberg, Inmmarie Hoeiden
    Affibody AB, Stockholm, Sweden..
    Gostring, Lovisa
    Affibody AB, Stockholm, Sweden..
    Uhlén, Mathias
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Hober, Sophia
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Selective enrichment of monospecific polyclonal antibodies for antibody-based proteomics efforts - Abstracts2004In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 3, no 10, p. S1-S1Article in journal (Other academic)
  • 18.
    Forsström, Bjorn
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Axnäs, Barbara Bislawska
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Stengele, Klaus-Peter
    Buehler, Jochen
    Albert, Thomas J.
    Richmond, Todd A.
    Hu, Francis Jingxin
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hudson, Elton Paul
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Rockberg, Johan
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Proteome-wide Epitope Mapping of Antibodies Using Ultra-dense Peptide Arrays2014In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 13, no 6, p. 1585-1597Article in journal (Refereed)
    Abstract [en]

    Antibodies are of importance for the field of proteomics, both as reagents for imaging cells, tissues, and organs and as capturing agents for affinity enrichment in mass-spectrometry-based techniques. It is important to gain basic insights regarding the binding sites (epitopes) of antibodies and potential cross-reactivity to nontarget proteins. Knowledge about an antibody's linear epitopes is also useful in, for instance, developing assays involving the capture of peptides obtained from trypsin cleavage of samples prior to mass spectrometry analysis. Here, we describe, for the first time, the design and use of peptide arrays covering all human proteins for the analysis of antibody specificity, based on parallel in situ photolithic synthesis of a total of 2.1 million overlapping peptides. This has allowed analysis of on-and off-target binding of both monoclonal and polyclonal antibodies, complemented with precise mapping of epitopes based on full amino acid substitution scans. The analysis suggests that linear epitopes are relatively short, confined to five to seven residues, resulting in apparent off-target binding to peptides corresponding to a large number of unrelated human proteins. However, subsequent analysis using recombinant proteins suggests that these linear epitopes have a strict conformational component, thus giving us new insights regarding how antibodies bind to their antigens.

  • 19. Geiger, T.
    et al.
    Velic, A.
    MacEk, B.
    Lundberg, E.
    Kampf, C.
    Nagaraj, N.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Cox, J.
    Mann, M.
    Initial quantitative proteomic map of 28 mouse tissues using the SILAC mouse2013In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 12, no 6, p. 1709-1722Article in journal (Refereed)
    Abstract [en]

    Identifying the building blocks of mammalian tissues is a precondition for understanding their function. In particular, global and quantitative analysis of the proteome of mammalian tissues would point to tissue-specific mechanisms and place the function of each protein in a whole-organism perspective. We performed proteomic analyses of 28 mouse tissues using high-resolution mass spectrometry and used a mix of mouse tissues labeled via stable isotope labeling with amino acids in cell culture as a "spike-in" internal standard for accurate protein quantification across these tissues. We identified a total of 7,349 proteins and quantified 6,974 of them. Bioinformatic data analysis showed that physiologically related tissues clustered together and that highly expressed proteins represented the characteristic tissue functions. Tissue specialization was reflected prominently in the proteomic profiles and is apparent already in their hundred most abundant proteins. The proportion of strictly tissue-specific proteins appeared to be small. However, even proteins with household functions, such as those in ribosomes and spliceosomes, can have dramatic expression differences among tissues. We describe a computational framework with which to correlate proteome profiles with physiological functions of the tissue. Our data will be useful to the broad scientific community as an initial atlas of protein expression of a mammalian species.

  • 20. Gloriam, David E.
    et al.
    Orchard, Sandra
    Bertinetti, Daniela
    Björling, Erik
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Bongcam-Rudloff, Erik
    Borrebaeck, Carl A. K.
    Bourbeillon, Julie
    Bradbury, Andrew R. M.
    de Daruvar, Antoine
    Duebel, Stefan
    Frank, Ronald
    Gibson, Toby J.
    Gold, Larry
    Haslam, Niall
    Herberg, Friedrich W.
    Hiltke, Tara
    Hoheisel, Joerg D.
    Kerrien, Samuel
    Koegl, Manfred
    Konthur, Zoltan
    Korn, Bernhard
    Landegren, Ulf
    Montecchi-Palazzi, Luisa
    Palcy, Sandrine
    Rodriguez, Henry
    Schweinsberg, Sonja
    Sievert, Volker
    Stoevesandt, Oda
    Taussig, Michael J.
    Ueffing, Marius
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    van der Maarel, Silvere
    Wingren, Christer
    Woollard, Peter
    Sherman, David J.
    Hermjakob, Henning
    A Community Standard Format for the Representation of Protein Affinity Reagents2010In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 9, no 1, p. 1-10Article in journal (Refereed)
    Abstract [en]

    Protein affinity reagents (PARs), most commonly antibodies, are essential reagents for protein characterization in basic research, biotechnology, and diagnostics as well as the fastest growing class of therapeutics. Large numbers of PARs are available commercially; however, their quality is often uncertain. In addition, currently available PARs cover only a fraction of the human proteome, and their cost is prohibitive for proteome scale applications. This situation has triggered several initiatives involving large scale generation and validation of antibodies, for example the Swedish Human Protein Atlas and the German Antibody Factory. Antibodies targeting specific subproteomes are being pursued by members of Human Proteome Organisation (plasma and liver proteome projects) and the United States National Cancer Institute (cancer-associated antigens). ProteomeBinders, a European consortium, aims to set up a resource of consistently quality-controlled protein-binding reagents for the whole human proteome. An ultimate PAR database resource would allow consumers to visit one online warehouse and find all available affinity reagents from different providers together with documentation that facilitates easy comparison of their cost and quality. However, in contrast to, for example, nucleotide databases among which data are synchronized between the major data providers, current PAR producers, quality control centers, and commercial companies all use incompatible formats, hindering data exchange. Here we propose Proteomics Standards Initiative (PSI)-PAR as a global community standard format for the representation and exchange of protein affinity reagent data. The PSI-PAR format is maintained by the Human Proteome Organisation PSI and was developed within the context of ProteomeBinders by building on a mature proteomics standard format, PSI-molecular interaction, which is a widely accepted and established community standard for molecular interaction data. Further information and documentation are available on the PSI-PAR web site. Molecular & Cellular Proteomics 9: 1-10, 2010.

  • 21.
    Hamsten, Carl
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Neiman, Maja
    KTH, School of Biotechnology (BIO), Proteomics.
    Schwenk, Jochen M.
    KTH, School of Biotechnology (BIO), Proteomics.
    Hamsten, Marica
    KTH, School of Biotechnology (BIO), Proteomics.
    March, John B.
    Persson, Anja
    KTH, School of Biotechnology (BIO), Proteomics.
    Recombinant surface proteomics as a tool to analyze humoral immune responses in bovines infected by Mycoplasma mycoides subsp. mycoides SC2009In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 8, no 11, p. 2544-2554Article in journal (Refereed)
    Abstract [en]

    A systematic approach to characterize the surface proteome of Mycoplasma mycoides subspecies mycoides small colony type (M. mycoides SC), the causing agent of contagious bovine pleuropneumonia (CBPP) in cattle, is presented. Humoral immune responses in 242 CBPP affected cattle and controls were monitored against one third of the surface proteins of M. mycoides SC in a high-throughput magnetic bead based assay. First, 64 surface proteins were selected from the genome sequence of M. mycoides SC and expressed as recombinant proteins in E. coli. Binding of antibodies to each individual protein could then be analyzed simultaneously in minute sample volumes with the Luminex suspension array technology. The assay was optimized on Namibian CBPP positive sera and Swedish negative controls to allow detection and 20-fold mean signal separation between CBPP positive and negative sera. Signals were proven to be protein-specific by inhibition experiments and results agreed with western blot experiments. The assay's potential to monitor IgG, IgM and IgA responses over time was shown in a proof-of-concept study with 116 sera from 8 animals in a CBPP vaccine study. In conclusion, a toolbox with recombinant proteins and a flexible suspension array assay that allows multiplex analysis of humoral immune responses to M mycoides SC, has been created.

  • 22. Janzi, M.
    et al.
    Ödling, Jenny
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Pan-Hammarstrom, Q.
    Sundberg, Mårten
    KTH, School of Biotechnology (BIO), Proteomics. KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics. KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Hammarstrom, L.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics. KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Serum microarrays for large scale screening of protein levels2005In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 4, no 12, p. 1942-1947Article in journal (Refereed)
    Abstract [en]

    There is a great need for comprehensive proteomic analysis of large patient cohorts of plasma and serum samples to identify biomarkers of human diseases. Here we describe a new antibody-based proteomic approach involving a reverse array format where serum samples are spotted on a microarray. This enables all samples to be screened for their content of a certain serum protein in a single experiment using target-recognizing antibodies and fluorescently labeled secondary antibodies. The procedure is illustrated with the analysis of the IgA levels in 2009 spotted serum samples, and the data are compared with clinical routine measurements. The results suggest that it is possible to simultaneously screen thousands of complex clinical serum samples for their content of the relative amount of specific serum proteins of clinical relevance.

  • 23.
    Kampf, C.
    et al.
    Uppsala Univ, Uppsala, Sweden..
    Bjorling, E.
    KTH.
    Wester, K.
    Uppsala Univ, Uppsala, Sweden..
    Andersson, A.
    Uppsala Univ, Uppsala, Sweden..
    Uhlén, Mathias
    KTH.
    Ponten, F.
    Uppsala Univ, Uppsala, Sweden..
    Mapping the human proteome using tissue microarrays2005In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 4, no 8, p. S63-S63Article in journal (Other academic)
  • 24. Lamond, Angus I.
    et al.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Horning, Stevan
    Makarov, Alexander
    Robinson, Carol V.
    Serrano, Luis
    Hartl, F. Ulrich
    Baumeister, Wolfgang
    Werenskiold, Anne Katrin
    Andersen, Jens S.
    Vorm, Ole
    Linial, Michal
    Aebersold, Ruedi
    Mann, Matthias
    Advancing Cell Biology Through Proteomics in Space and Time (PROSPECTS)2012In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 11, no 3Article in journal (Refereed)
    Abstract [en]

    The term "proteomics" encompasses the large-scale detection and analysis of proteins and their post-translational modifications. Driven by major improvements in mass spectrometric instrumentation, methodology, and data analysis, the proteomics field has burgeoned in recent years. It now provides a range of sensitive and quantitative approaches for measuring protein structures and dynamics that promise to revolutionize our understanding of cell biology and molecular mechanisms in both human cells and model organisms. The Proteomics Specification in Time and Space (PROSPECTS) Network is a unique EU-funded project that brings together leading European research groups, spanning from instrumentation to biomedicine, in a collaborative five year initiative to develop new methods and applications for the functional analysis of cellular proteins. This special issue of Molecular and Cellular Proteomics presents 16 research papers reporting major recent progress by the PROSPECTS groups, including improvements to the resolution and sensitivity of the Orbitrap family of mass spectrometers, systematic detection of proteins using highly characterized antibody collections, and new methods for absolute as well as relative quantification of protein levels. Manuscripts in this issue exemplify approaches for performing quantitative measurements of cell proteomes and for studying their dynamic responses to perturbation, both during normal cellular responses and in disease mechanisms. Here we present a perspective on how the proteomics field is moving beyond simply identifying proteins with high sensitivity toward providing a powerful and versatile set of assay systems for characterizing proteome dynamics and thereby creating a new "third generation"proteomics strategy that offers an indispensible tool for cell biology and molecular medicine.

  • 25. Larance, Mark
    et al.
    Kirkwood, Kathryn J.
    Xirodimas, Dimitris P.
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lamond, Angus I.
    Characterization of MRFAP1 Turnover and Interactions Downstream of the NEDD8 Pathway2012In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 11, no 3Article in journal (Refereed)
    Abstract [en]

    The NEDD8-Cullin E3 ligase pathway plays an important role in protein homeostasis, in particular the degradation of cell cycle regulators and transcriptional control networks. To characterize NEDD8-cullin target proteins, we performed a quantitative proteomic analysis of cells treated with MLN4924, a small molecule inhibitor of the NEDD8 conjugation pathway. MRFAP1 and its interaction partner, MORF4L1, were among the most up-regulated proteins after NEDD8 inhibition in multiple human cell lines. We show that MRFAP1 has a fast turnover rate in the absence of MLN4924 and is degraded via the ubiquitin- proteasome system. The increased abundance of MRFAP1 after MLN4924 treatment results from a decreased rate of degradation. Characterization of the binding partners of both MRFAP1 and MORF4L1 revealed a complex protein-protein interaction network. MRFAP1 bound to a number of E3 ubiquitin ligases, including CUL4B, but not to components of the NuA4 complex, including MRGBP, which bound to MORF4L1. These data indicate that MRFAP1 may regulate the ability of MORF4L1 to interact with chromatin-modifying enzymes by binding to MORF4L1 in a mutually exclusive manner with MRGBP. Analysis of MRFAP1 expression in human tissues by immunostaining with a MRFAP1-specific antibody revealed that it was detectable in only a small number of tissues, in particular testis and brain. Strikingly, analysis of the seminiferous tubules of the testis showed the highest nuclear staining in the spermatogonia and much weaker staining in the spermatocytes and spermatids. MRGBP was inversely correlated with MRFAP1 expression in these cell types, consistent with an exchange of MORF4L1 interaction partners as cells progress through meiosis in the testis. These data highlight an important new arm of the NEDD8cullin pathway.

  • 26.
    Lee, Chien-Yun
    et al.
    Tech Univ Munich, Chair Prote & Bioanalyt, Emil Erlenmeyer Forum 5, D-85354 Freising Weihenstephan, Germany.;Natl Chung Hsing Univ, Grad Inst Biotechnol, Taichung, Taiwan.;Acad Sinica, Mol & Biol Agr Sci Program, Taiwan Int Grad Program, Taipei, Taiwan.;Natl Chung Hsing Univ, Taipei, Taiwan..
    Wang, Dongxue
    Tech Univ Munich, Chair Prote & Bioanalyt, Emil Erlenmeyer Forum 5, D-85354 Freising Weihenstephan, Germany..
    Wilhelm, Mathias
    Tech Univ Munich, Chair Prote & Bioanalyt, Emil Erlenmeyer Forum 5, D-85354 Freising Weihenstephan, Germany..
    Zolg, Daniel P.
    Tech Univ Munich, Chair Prote & Bioanalyt, Emil Erlenmeyer Forum 5, D-85354 Freising Weihenstephan, Germany..
    Schmidt, Tobias
    Tech Univ Munich, Chair Prote & Bioanalyt, Emil Erlenmeyer Forum 5, D-85354 Freising Weihenstephan, Germany..
    Schnatbaum, Karsten
    JPT Peptide Technol GmbH, Berlin, Germany..
    Reimer, Ulf
    JPT Peptide Technol GmbH, Berlin, Germany..
    Ponten, Fredrik
    Uppsala Univ, Dept Immunol Genet & Pathol, Uppsala, Sweden..
    Uhlén, Mathias
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hahne, Hannes
    OmicScouts GmbH, Freising Weihenstephan, Germany..
    Kuster, Bernhard
    Tech Univ Munich, Chair Prote & Bioanalyt, Emil Erlenmeyer Forum 5, D-85354 Freising Weihenstephan, Germany.;Natl Chung Hsing Univ, Taipei, Taiwan.;Bavarian Ctr Biomol Mass Spectrometry, Freising Weihenstephan, Germany..
    Mining the Human Tissue Proteome for Protein Citrullination2018In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 17, no 7, p. 1378-1391Article in journal (Refereed)
    Abstract [en]

    Citrullination is a posttranslational modification of arginine catalyzed by five peptidylarginine deiminases (PADs) in humans. The loss of a positive charge may cause structural or functional alterations, and while the modification has been linked to several diseases, including rheumatoid arthritis (RA) and cancer, its physiological or pathophysiological roles remain largely unclear. In part, this is owing to limitations in available methodology to robustly enrich, detect, and localize the modification. As a result, only a few citrullination sites have been identified on human proteins with high confidence. In this study, we mined data from mass-spectrometry-based deep proteomic profiling of 30 human tissues to identify citrullination sites on endogenous proteins. Database searching of similar to 70 million tandem mass spectra yielded similar to 13,000 candidate spectra, which were further triaged by spectrum quality metrics and the detection of the specific neutral loss of isocyanic acid from citrullinated peptides to reduce false positives. Because citrullination is easily confused with deamidation, we synthetized similar to 2,200 citrullinated and 1,300 deamidated peptides to build a library of reference spectra. This led to the validation of 375 citrullination sites on 209 human proteins. Further analysis showed that >80% of the identified modifications sites were new, and for 56% of the proteins, citrullination was detected for the first time. Sequence motif analysis revealed a strong preference for Asp and Gly, residues around the citrullination site. Interestingly, while the modification was detected in 26 human tissues with the highest levels found in the brain and lung, citrullination levels did not correlate well with protein expression of the PAD enzymes. Even though the current work represents the largest survey of protein citrullination to date, the modification was mostly detected on high abundant proteins, arguing that the development of specific enrichment methods would be required in order to study the full extent of cellular protein citrullination.

  • 27. Legrain, Pierre
    et al.
    Aebersold, Ruedi
    Archakov, Alexander
    Bairoch, Amos
    Bala, Kumar
    Beretta, Laura
    Bergeron, John
    Borchers, Christoph H.
    Corthals, Garry L.
    Costello, Catherine E.
    Deutsch, Eric W.
    Domon, Bruno
    Hancock, William
    He, Fuchu
    Hochstrasser, Denis
    Marko-Varga, Gyorgy
    Salekdeh, Ghasem Hosseini
    Sechi, Salvatore
    Snyder, Michael
    Srivastava, Sudhir
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Wu, Cathy H.
    Yamamoto, Tadashi
    Paik, Young-Ki
    Omenn, Gilbert S.
    The Human Proteome Project: Current State and Future Direction2011In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 10, no 7Article in journal (Refereed)
    Abstract [en]

    After the successful completion of the Human Genome Project, the Human Proteome Organization has recently officially launched a global Human Proteome Project (HPP), which is designed to map the entire human protein set. Given the lack of protein-level evidence for about 30% of the estimated 20,300 protein-coding genes, a systematic global effort will be necessary to achieve this goal with respect to protein abundance, distribution, subcellular localization, interaction with other biomolecules, and functions at specific time points. As a general experimental strategy, HPP research groups will use the three working pillars for HPP: mass spectrometry, antibody capture, and bioinformatics tools and knowledge bases. The HPP participants will take advantage of the output and cross-analyses from the ongoing Human Proteome Organization initiatives and a chromosome-centric protein mapping strategy, termed C-HPP, with which many national teams are currently engaged. In addition, numerous biologically driven and disease-oriented projects will be stimulated and facilitated by the HPP. Timely planning with proper governance of HPP will deliver a protein parts list, reagents, and tools for protein studies and analyses, and a stronger basis for personalized medicine. The Human Proteome Organization urges each national research funding agency and the scientific community at large to identify their preferred pathways to participate in aspects of this highly promising project in a HPP consortium of funders and investigators.

  • 28.
    Lindskog, M.
    et al.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Berglund, L.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Sterky, Fredrik
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Selection of antigenic protein fragments for antibody-based proteomics2005In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 4, no 8, p. S63-S63Article in journal (Other academic)
  • 29.
    Lundberg, E.
    et al.
    KTH.
    Sundberg, M.
    KTH.
    Gräslund, Torbjörn
    KTH.
    Uhlén, Mathias
    KTH.
    Brismar, Hjalmar
    KTH.
    Andersson, H.
    KTH.
    Development of a platform for highthroughput quality assurance of monospecific polyclonal antibodies based on confocal scanning laser microscopy and FRET analysis2006In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 5, no 10, p. S222-S222Article in journal (Other academic)
  • 30. Mulder, J.
    et al.
    Björling, Erik
    KTH, School of Biotechnology (BIO), Proteomics.
    Jonasson, Kalle
    KTH, School of Biotechnology (BIO), Proteomics.
    Wernérus, Henrik
    KTH, School of Biotechnology (BIO), Proteomics.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Hokfelt, T.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Tissue Profiling of the Mammalian Central Nervous System Using Human Antibody-based Proteomics2009In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 8, no 7, p. 1612-1622Article in journal (Refereed)
    Abstract [en]

    A need exists for mapping the protein profiles in the human brain both during normal and disease conditions. Here we studied 800 antibodies generated toward human proteins as part of a Human Protein Atlas program and investigated their suitability for detailed analysis of various levels of a rat brain using immuno-based methods. In this way, the parallel, rather limited analysis of the human brain, restricted to four brain areas (cerebellum, cerebral cortex, hippocampus, and lateral subventricular zone), could be extended in the rat model to 25 selected areas of the brain. Approximately 100 antibodies (12%) revealed a distinct staining pattern and passed validation of specificity using Western blot analysis. These antibodies were applied to coronal sections of the rat brain at 0.7-mm intervals covering the entire brain. We have now produced detailed protein distribution profiles for these antibodies and acquired over 640 images that form the basis of a publicly available portal of an antibody-based Rodent Brain Protein Atlas database (www.proteinatlas.org/rodentbrain). Because of the systematic selection of target genes, the majority of antibodies included in this database are generated against proteins that have not been studied in the brain before. Furthermore optimized tissue processing and colchicine treatment allow a high quality, more extended annotation and detailed analysis of subcellular distributions and protein dynamics. Molecular & Cellular Proteomics 8: 1612-1622, 2009.

  • 31. Nilsson, P.
    et al.
    Bjorklund, M. G.
    Asplund, A.
    Rimini, R.
    Stromberg, S.
    Uhlen, M.
    KTH.
    Ponten, F.
    Large scale comparative mRNA and protein expression profiling2006In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 5, no 10, p. S184-S184Article in journal (Other academic)
  • 32.
    Nilsson, P.
    et al.
    KTH.
    Janzi, M.
    KTH.
    Odling, J.
    KTH.
    Sundberg, M.
    KTH.
    Lundeberg, Joakim
    KTH.
    Uhlén, Mathias
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Hammarstrom, L.
    KTH.
    Serum microarrays for screening of protein levels2005In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 4, no 8, p. S334-S334Article in journal (Other academic)
  • 33.
    Ottosson, J.
    et al.
    KTH.
    Steen, J.
    Tegel, Hanna
    KTH.
    Konrad, A.
    KTH.
    Halimi, A.
    KTH.
    Wrethagen, U.
    KTH.
    Xu, L. Lan
    KTH.
    Pettersson, K.
    KTH.
    Widehammar, J.
    KTH.
    Dahlgren, L. -G
    KTH.
    Hober, Sophia
    KTH.
    High throughput protein production and purification in the Human Protein Atlas program2006In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 5, no 10, p. S40-S40Article in journal (Other academic)
  • 34.
    Ottosson, J.
    et al.
    KTH.
    Wernerus, H.
    KTH.
    Nilsson, P.
    KTH.
    Tegel, Hanna
    KTH.
    Larsson, K.
    KTH.
    Uhlén, Mathias
    KTH.
    Hober, S.
    KTH.
    High throughput antibody generation and validation for antibody proteomics2005In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 4, no 8, p. S64-S64Article in journal (Other academic)
  • 35.
    Ottosson, Jenny
    et al.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Steen, Johanna
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Stenvall, Maria
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Tegel, Hanna
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Hober, Sophia
    KTH, Superseded Departments (pre-2005), Biotechnology.
    High throughput protein expression and purification for antibody proteomics2004In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 3, no 10, p. S169-S169Article in journal (Other academic)
  • 36.
    Persson, A.
    et al.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Tourle, S.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Ekström, M.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Amini, B.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Skollermo, A.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Sundberg, M.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Westberg, J.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Ottosson, J.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Al-Khalili Szigyarto, Cristina
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments (pre-2005), Biotechnology.
    High throughput cloning and production of protein epitope signature tags from genes on chromosome 14, 22, X, and Y2004In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 3, no 10, p. S169-S169Article in journal (Other academic)
  • 37. Persson, B. S.
    et al.
    Sundberg, M.
    Nilsson, P.
    Specificity validation of mono-specific antibodies by label-free interaction analysis using a protein interaction array2006In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 5, no 10, p. S66-S66Article in journal (Other academic)
  • 38. Poetz, Oliver
    et al.
    Henzler, Tanja
    Hartmann, Michael
    KTH, School of Chemical Science and Engineering (CHE), Chemistry.
    Kazmaier, Cornelia
    Templin, Markus F.
    Herget, Thomas
    Joos, Thomas O.
    Sequential Multiplex Analyte Capturing for Phosphoprotein Profiling2010In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 9, no 11, p. 2474-2481Article in journal (Refereed)
    Abstract [en]

    Microarray-based sandwich immunoassays can simultaneously detect dozens of proteins. However, their use in quantifying large numbers of proteins is hampered by cross-reactivity and incompatibilities caused by the immunoassays themselves. Sequential multiplex analyte capturing addresses these problems by repeatedly probing the same sample with different sets of antibodycoated, magnetic suspension bead arrays. As a miniaturized immunoassay format, suspension bead array-based assays fulfill the criteria of the ambient analyte theory, and our experiments reveal that the analyte concentrations are not significantly changed. The value of sequential multiplex analyte capturing was demonstrated by probing tumor cell line lysates for the abundance of seven different receptor tyrosine kinases and their degree of phosphorylation and by measuring the complex phosphorylation pattern of the epidermal growth factor receptor in the same sample from the same cavity. Molecular & Cellular Proteomics 9:2474-2481, 2010.

  • 39.
    Ponten, F.
    et al.
    Rudbeck Lab, IGP, Uppsala, Sweden..
    Kampf, C.
    Rudbeck Lab, IGP, Uppsala, Sweden..
    Wester, K.
    Rudbeck Lab, IGP, Uppsala, Sweden..
    Andersson, A.
    Rudbeck Lab, IGP, Uppsala, Sweden..
    Bjorling, E.
    Rudbeck Lab, IGP, Uppsala, Sweden..
    Uhlén, Mathias
    KTH.
    Antibody-based proteomics for human tissue profiling; the Swedish Human Proteome Resource project (HPR)2005In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 4, no 8, p. S65-S65Article in journal (Other academic)
  • 40.
    Rockberg, Johan
    et al.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Szigyarto, Cristina AI-Khalili
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Antigen selection for design of protein epitope signature tags2004In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 3, no 10, p. S5-S5Article in journal (Other academic)
  • 41.
    Schwenk, Jochen M.
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Igel, Ulrika
    KTH, School of Biotechnology (BIO), Proteomics.
    Neiman, Maja
    KTH, School of Biotechnology (BIO), Proteomics.
    Langen, Hanno
    Becker, Charlotte
    Bjartell, Anders
    Ponten, Fredrik
    Wiklund, Fredrik
    Gronberg, Henrik
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Toward Next Generation Plasma Profiling via Heat-induced Epitope Retrieval and Array-based Assays2010In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 9, no 11, p. 2497-2507Article in journal (Refereed)
    Abstract [en]

    There is a need for high throughput methods for screening patient samples in the quest for potential biomarkers for diagnostics and patient care. Here, we used a combination of undirected target selection, antibody suspension bead arrays, and heat-induced epitope retrieval to allow for protein profiling of human plasma in a novel and systematic manner. Several antibodies were found to reveal altered protein profiles upon epitope retrieval at elevated temperatures with limits of detection improving into lower ng/ml ranges. In a study based on prostate cancer patients, several proteins with differential profiles were discovered and subsequently validated in an independent cohort. For one of the potential biomarkers, the human carnosine dipeptidase 1 protein (CNDP1), the differences were determined to be related to the glycosylation status of the targeted protein. The study shows a path of pursuit for large scale screening of biobank repositories in a flexible and proteome-wide fashion by utilizing heat-induced epitope retrieval and using an antibody suspension bead array format. Molecular & Cellular Proteomics 9:2497-2507, 2010.

  • 42.
    Schwenk, Jochen M.
    et al.
    KTH, School of Biotechnology (BIO).
    Lindberg, Johan
    Sundberg, Mårten
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Determination of binding specificities in highly multiplexed bead-based assays for antibody proteomics2007In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 6, no 1, p. 125-132Article in journal (Refereed)
    Abstract [en]

    One of the major challenges of antibody-based proteomics is the quality assurance of the generated antibodies to ensure specificity to the target protein. Here we describe a single tube multiplex approach to simultaneously analyze the binding of antibodies to a large number of different antigens. This bead-based assay utilizes the full multiplexing capacity theoretically offered by the Luminex suspension array technology. A protocol for an increased coupling throughput for the immobilization of antigens was developed and used to set up complex and stabile 100-plex bead mixtures. The possibility of using a two-dimensional multiplexing, in terms of high numbers of both analytes and samples or as in this case antigens and antibodies, enables the specificity of 96 antibodies versus 100 different antigens to be determined in 2 h. This high throughput analysis will potentially have great impact on the possibility for the utilization of different antibody proteomics approaches where the quality assessment of antibodies is of the utmost importance.

  • 43.
    Srivastava, Vaibhav
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Malm, Erik
    KTH, School of Biotechnology (BIO), Glycoscience.
    Sundqvist, Gustav
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Quantitative Proteomics Reveals that Plasma Membrane Microdomains From Poplar Cell Suspension Cultures Are Enriched in Markers of Signal Transduction, Molecular Transport, and Callose Biosynthesis2013In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 12, no 12, p. 3874-3885Article in journal (Refereed)
    Abstract [en]

    The plasma membrane (PM) is a highly dynamic interface that contains detergent-resistant microdomains (DRMs). The aim of this work was to determine the main functions of such microdomains in poplar through a proteomic analysis using gel-based and solution (iTRAQ) approaches. A total of 80 proteins from a limited number of functional classes were found to be significantly enriched in DRM relative to PM. The enriched proteins are markers of signal transduction, molecular transport at the PM, or cell wall biosynthesis. Their intrinsic properties are presented and discussed together with the biological significance of their enrichment in DRM. Of particular importance is the significant and specific enrichment of several callose [(1→3)-β-glucan] synthase isoforms, whose catalytic activity represents a final response to stress, leading to the deposition of callose plugs at the surface of the PM. An integrated functional model that connects all DRM-enriched proteins identified is proposed. This report is the only quantitative analysis available to date of the protein composition of membrane microdomains from a tree species.

  • 44.
    Sterky, F.
    KTH.
    Characterization of regulatory genes in the secondary meristem of populus by in situ protein localization2005In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 4, no 8, p. S260-S260Article in journal (Other academic)
  • 45.
    Sterky, Fredrik
    et al.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Berglund, L.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Lindskog, M.
    KTH, School of Biotechnology (BIO).
    Rockberg, Johan
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Al-Khalili Szigyarto, Cristina
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Strategies and software for design of protein Epitopes within HPR2004In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 3, no 10, p. S4-S4Article in journal (Other academic)
  • 46.
    Szigyarto, C. Al-Khalili
    et al.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Berglund, L.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Sivertsson, Åsa
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Lindskog, M.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Rockberg, J.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Westberg, J.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Agaton, L.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Persson, A.
    KTH, Superseded Departments (pre-2005), Biotechnology. Royal Inst Technol, Dept Mol Biotechnol, Stockholm, Sweden..
    Uhlén, Mathias
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Statistics of protein epitope signature tag design within the Swedish human proteom resource2004In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 3, no 10, p. S91-S91Article in journal (Other academic)
  • 47.
    Tegel, Hanna
    et al.
    KTH.
    Hedhammar, My
    KTH.
    Uhlén, Mathias
    KTH.
    Ottosson, J.
    KTH.
    Hober, Sophia
    KTH.
    Novel flow cytometry-based method for analysis of protein production in Escherichia coli2005In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 4, no 8, p. S66-S66Article in journal (Other academic)
  • 48. Ting, Ying S.
    et al.
    Egertson, Jarrett D.
    Payne, Samuel H.
    Kim, Sangtae
    MacLean, Brendan
    Käll, Lukas
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Aebersold, Ruedi
    Smith, Richard D.
    Noble, William Stafford
    MacCoss, Michael J.
    Peptide-Centric Proteome Analysis: An Alternative Strategy for the Analysis of Tandem Mass Spectrometry Data2015In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 14, no 9, p. 2301-2307Article, review/survey (Refereed)
    Abstract [en]

    In mass spectrometry-based bottom-up proteomics, data-independent acquisition is an emerging technique because of its comprehensive and unbiased sampling of precursor ions. However, current data-independent acquisition methods use wide precursor isolation windows, resulting in cofragmentation and complex mixture spectra. Thus, conventional database searching tools that identify peptides by interpreting individual tandem MS spectra are inherently limited in analyzing data-independent acquisition data. Here we discuss an alternative approach, peptide-centric analysis, which tests directly for the presence and absence of query peptides. We discuss how peptide-centric analysis resolves some limitations of traditional spectrum-centric analysis, and we outline the unique characteristics of peptide-centric analysis in general.

  • 49.
    Tourle, S.
    et al.
    KTH.
    Tegel, Hanna
    KTH.
    Ottosson, J.
    KTH.
    Persson, A.
    KTH.
    Increased levels of recombinant human proteins in E-Coli Rosetta that compensates for mammalian codon usage2006In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 5, no 10, p. S223-S223Article in journal (Other academic)
  • 50.
    Uhlén, Mathias
    KTH.
    A human protein atlas for expression profiles in human tissues and cells2006In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 5, no 10, p. S378-S378Article in journal (Other academic)
12 1 - 50 of 60
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