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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    KTH, Superseded Departments, Biotechnology.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    KTH, Superseded Departments, Biotechnology.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    KTH, Superseded Departments, Biotechnology.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.
    Apraiz, Itxaso
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mi, Jia
    Cristobal, Susana
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Identification of proteomic signatures of exposure to marine pollutants in mussels (Mytilus edulis)2006In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 5, no 7, p. 1274-1285Article in journal (Refereed)
  • 5.
    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)
  • 6.
    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.

  • 7. Baan, Bart
    et al.
    Pardali, Evangelia
    ten Dijke, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    van Dam, Hans
    In situ proximity ligation detection of c-Jun/AP-1 dimers reveals increased levels of c-Jun/Fra1 complexes in aggressive breast cancer cell lines in vitro and in vivo2010In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 9, no 9, p. 1982-1990Article in journal (Refereed)
    Abstract [en]

    Genetic and biochemical studies have shown that selective interactions between the Jun, Fos, and activating transcription factor (ATF) components of transcription factor activating protein 1 (AP-1) exhibit specific and critical functions in the regulation of cell proliferation, differentiation, and survival. For instance, the ratio between c-Jun/c-Fos and c-Jun/ATF2 dimers in the cell can be a determining factor in the cellular response to oncogenic or apoptotic stimuli. Until recently, no methods were available to detect endogenous AP-1 complexes in cells and tissues in situ. Here, we validated the proximity ligation assay (PLA) for its ability to specifically visualize and quantify changes in endogenous c-Jun/c-Fos, c-Jun/ATF2, and c-Jun/Fra1 complexes by using, among others, partner-selective c-Jun mutants. Furthermore, we examined the levels of c-Jun/AP-1 dimers in cell lines representing different types of human breast cancer and found that aggressive basal-like breast cancer cells can be discriminated from much less invasive luminal-like cells by PLA detection of c-Jun/Fra1 rather than of c-Jun/ATF2 and c-Jun/c-Fos. Also in tumor tissue derived from highly metastatic basal-like MDA-MB231 cells, high levels of c-Jun/Fra1 complexes were detected. Together, these results demonstrate that in situ PLA is a powerful diagnostic tool to analyze and quantify the amounts of biologically critical AP-1 dimers in fixed cells and tissue material.

  • 8. Balonova, Lucie
    et al.
    Mann, Benjamin F
    Cerveny, Lukas
    Alley, William R, Jr
    Chovancova, Eva
    Forslund, Anna-Lena
    Salomonsson, Emelie N
    Forsberg, Åke
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Damborsky, Jiri
    Novotny, Milos V
    Hernychova, Lenka
    Stulik, Jiri
    Characterization of protein glycosylation in Francisella tularensis subsp holarctica2012In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 11, no 7Article in journal (Refereed)
    Abstract [en]

    FTH_0069 is a previously uncharacterized strongly immunoreactive protein that has been proposed to be a novel virulence factor in Francisella tularensis. Here, the glycan structure modifying two C-terminal peptides of FTH_0069 was identified utilizing high resolution, high mass accuracy mass spectrometry, combined with in-source CID tandem MS experiments. The glycan observed at m/z 1156 was determined to be a hexasaccharide, consisting of two hexoses, three N-acetylhexosamines, and an unknown monosaccharide containing a phosphate group. The monosaccharide sequence of the glycan is tentatively proposed as X-P-HexNAc-HexNAc-Hex-Hex-HexNAc, where X denotes the unknown monosaccharide. The glycan is identical to that of DsbA glycoprotein, as well as to one of the multiple glycan structures modifying the type IV pilin PilA, suggesting a common biosynthetic pathway for the protein modification. Here, we demonstrate that the glycosylation of FTH_0069, DsbA, and PilA was affected in an isogenic mutant with a disrupted wbtDEF gene cluster encoding O-antigen synthesis and in a mutant with a deleted pglA gene encoding pilin oligosaccharyltransferase PglA. Based on our findings, we propose that PglA is involved in both pilin and general F. tularensis protein glycosylation, and we further suggest an inter-relationship between the O-antigen and the glycan synthesis in the early steps in their biosynthetic pathways. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.015016, 1-12, 2012.

  • 9.
    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)
  • 10. Barbe, Laurent
    et al.
    Lundberg, Emma
    Oksvold, Per
    Stenius, Anna
    Lewin, Erland
    Björling, Erik
    Asplund, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Pontén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Brismar, Hjalmar
    Uhlén, Mathias
    Andersson-Svahn, Helene
    Toward a confocal subcellular atlas of the human proteome2008In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 7, no 3, p. 499-508Article in journal (Refereed)
    Abstract [en]

    Information on protein localization on the subcellular level is important to map and characterize the proteome and to better understand cellular functions of proteins. Here we report on a pilot study of 466 proteins in three human cell lines aimed to allow large scale confocal microscopy analysis using protein-specific antibodies. Approximately 3000 high resolution images were generated, and more than 80% of the analyzed proteins could be classified in one or multiple subcellular compartment(s). The localizations of the proteins showed, in many cases, good agreement with the Gene Ontology localization prediction model. This is the first large scale antibody-based study to localize proteins into subcellular compartments using antibodies and confocal microscopy. The results suggest that this approach might be a valuable tool in conjunction with predictive models for protein localization.

  • 11. Berggrund, Malin
    et al.
    Enroth, Stefan
    Lundberg, Martin
    Assarsson, Erika
    Stålberg, Karin
    Lindquist, David
    Umeå University, Faculty of Medicine, Department of Radiation Sciences.
    Hallmans, Göran
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Nutritional Research.
    Grankvist, Kjell
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Clinical chemistry.
    Olovsson, Matts
    Gyllensten, Ulf
    Identification of candidate plasma protein biomarkers for cervical cancer using the multiplex proximity extension assay2019In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 18, no 4, p. 735-743, article id RA118.001208Article in journal (Refereed)
    Abstract [en]

    Human papillomavirus (HPV) is recommended as the primary test in cervical cancer screening, with co-testing by cytology for HPV-positive women to identify cervical lesions. Cytology has low sensitivity and there is a need to identify biomarkers that could identify dysplasia that are likely to progress to cancer. We searched for plasma proteins that could identify women with cervical cancer using the multiplex proximity extension assay (PEA). The abundance of 100 proteins were measured in plasma collected at the time of diagnosis of patients with invasive cervical cancer and in population controls using the Olink Multiplex panels CVD II, INF I, and ONC II. Eighty proteins showed increased levels in cases compared to controls. We identified a signature of 11 proteins (PTX3, ITGB1BP2, AXIN1, STAMPB, SRC, SIRT2, 4E-BP1, PAPPA, HB-EGF, NEMO and IL27) that distinguished cases and controls with a sensitivity of 0.96 at a specificity of 1.0. This signature was evaluated in a prospective replication cohort with samples collected before, at or after diagnosis and achieved a sensitivity of 0.78 and a specificity 0.56 separating samples collected at the time of diagnosis of invasive cancer from samples collected prior to diagnosis. No difference in abundance was seen between samples collected prior to diagnosis or after treatment as compared to population controls, indicating that this protein signature is mainly informative close to time of diagnosis. Further studies are needed to determine the optimal window in time prior to diagnosis for these biomarker candidates.

  • 12.
    Berggrund, Malin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Enroth, Stefan
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik.
    Lundberg, Martin
    OLINK Prote, Uppsala Sci Pk, SE-75183 Uppsala, Sweden.
    Assarsson, Erika
    OLINK Prote, Uppsala Sci Pk, SE-75183 Uppsala, Sweden.
    Stålberg, Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health, Research group (Dept. of women´s and children´s health), Reproductive Health.
    Lindquist, David
    Umeå Univ, Dept Radiat Sci, SE-90187 Umeå, Sweden.
    Hallmans, Göran
    Umeå Univ, Dept Publ Hlth & Clin Med, Nutr Res, SE-90187 Umeå, Sweden.
    Grankvist, Kjell
    Umeå Univ, Dept Med Biosci, Clin Chem, SE-90187 Umeå, Sweden.
    Olovsson, Matts
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health, Research group (Dept. of women´s and children´s health), Reproductive biology.
    Gyllensten, Ulf
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik.
    Identification of Candidate Plasma Protein Biomarkers for Cervical Cancer Using the Multiplex Proximity Extension Assay2019In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 18, no 4, p. 735-743Article in journal (Refereed)
    Abstract [en]

    Human papillomavirus (HPV) is recommended as the primary test in cervical cancer screening, with co-testing by cytology for HPV-positive women to identify cervical lesions. Cytology has low sensitivity and there is a need to identify biomarkers that could identify dysplasia that are likely to progress to cancer. We searched for plasma proteins that could identify women with cervical cancer using the multiplex proximity extension assay (PEA). The abundance of 100 proteins were measured in plasma collected at the time of diagnosis of patients with invasive cervical cancer and in population controls using the Olink Multiplex panels CVD II, INF I, and ONC II. Eighty proteins showed increased levels in cases compared with controls. We identified a signature of 11 proteins (PTX3, ITGB1BP2, AXIN1, STAMPB, SRC, SIRT2, 4E-BP1, PAPPA, HB-EGF, NEMO and IL27) that distinguished cases and controls with a sensitivity of 0.96 at a specificity of 1.0. This signature was evaluated in a prospective replication cohort with samples collected before, at or after diagnosis and achieved a sensitivity of 0.78 and a specificity 0.56 separating samples collected at the time of diagnosis of invasive cancer from samples collected prior to diagnosis. No difference in abundance was seen between samples collected prior to diagnosis or after treatment as compared with population controls, indicating that this protein signature is mainly informative close to time of diagnosis. Further studies are needed to determine the optimal window in time prior to diagnosis for these biomarker candidates.

  • 13. Berglund, Lisa
    et al.
    Björling, Erik
    Oksvold, Per
    Fagerberg, Linn
    Asplund, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Szigyarto, Cristina Al-Khalili
    Persson, Anja
    Ottosson, Jenny
    Wernérus, Henrik
    Nilsson, Peter
    Lundberg, Emma
    Sivertsson, Åsa
    Navani, Sanjay
    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.
    Hober, Sophia
    Pontén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Uhlén, Mathias
    A genecentric Human Protein Atlas for expression profiles based on antibodies2008In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 7, no 10, p. 2019-2027Article, review/survey (Refereed)
    Abstract [en]

    An attractive path forward in proteomics is to experimentally annotate the human protein complement of the genome in a genecentric manner. Using antibodies, it might be possible to design protein-specific probes for a representative protein from every protein-coding gene and to subsequently use the antibodies for systematical analysis of cellular distribution and subcellular localization of proteins in normal and disease tissues. A new version (4.0) of the Human Protein Atlas has been developed in a genecentric manner with the inclusion of all human genes and splice variants predicted from genome efforts together with a visualization of each protein with characteristics such as predicted membrane regions, signal peptide, and protein domains and new plots showing the uniqueness (sequence similarity) of every fraction of each protein toward all other human proteins. The new version is based on tissue profiles generated from 6120 antibodies with more than five million immunohistochemistry-based images covering 5067 human genes, corresponding to approximately 25% of the human genome. Version 4.0 includes a putative list of members in various protein classes, both functional classes, such as kinases, transcription factors, G-protein-coupled receptors, etc., and project-related classes, such as candidate genes for cancer or cardiovascular diseases. The exact antigen sequence for the internally generated antibodies has also been released together with a visualization of the application-specific validation performed for each antibody, including a protein array assay, Western blot analysis, immunohistochemistry, and, for a large fraction, immunofluorescence-based confocal microscopy. New search functionalities have been added to allow complex queries regarding protein expression profiles, protein classes, and chromosome location. The new version of the protein atlas thus is a resource for many areas of biomedical research, including protein science and biomarker discovery.

  • 14. 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)
  • 15.
    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)
  • 16.
    Björkesten, Johan
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Enroth, Stefan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Shen, Qiujin
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Wik, Lotta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hougaard, David
    Statens Serum Inst, Danish Ctr Neonatal Screening, Copenhagen, Denmark.
    Cohen, Arieh
    Statens Serum Inst, Danish Ctr Neonatal Screening, Copenhagen, Denmark.
    Sörensen, Lene
    Karolinska Univ Hosp, Ctr Inherited Metab Dis, Stockholm, Sweden.
    Giedraitis, Vilmantas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Ingelsson, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Larsson, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Stability of Proteins in Dried Blood Spot Biobanks.2017In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 16, no 7, p. 1286-1296Article in journal (Refereed)
    Abstract [en]

    An important motivation for the construction of biobanks is to discover biomarkers that identify diseases at early, potentially curable stages. This will require biobanks from large numbers of individuals, preferably sampled repeatedly, where the samples are collected and stored under conditions that preserve potential biomarkers. Dried blood samples are attractive for biobanking because of the ease and low cost of collection and storage. Here we have investigated their suitability for protein measurements. 92 proteins with relevance for oncology were analyzed using multiplex proximity extension assays (PEA) in dried blood spots collected on paper and stored for up to 30 years at either +4&deg;C or -24&deg;C.</p> <p>Our main findings were that 1) the act of drying only slightly influenced detection of blood proteins (average correlation of 0.970), and in a reproducible manner (correlation of 0.999), 2) detection of some proteins was not significantly affected by storage over the full range of three decades (34% and 76% of the analyzed proteins at +4&deg;C and -24&deg;C, respectively), while levels of others decreased slowly during storage with half-lives in the range of 10 to 50 years, and 3) detectability of proteins was less affected in dried samples stored at -24&deg;C compared to at +4&deg;C, as the median protein abundance had decreased to 80% and 93% of starting levels after 10 years of storage at +4&deg;C or -24&deg;C, respectively. The results of our study are encouraging as they suggest an inexpensive means to collect large numbers of blood samples, even by the donors themselves, and to transport, and store biobanked samples as spots of whole blood dried on paper. Combined with emerging means to measure hundreds or thousands of protein, such biobanks could prove of great medical value by greatly enhancing discovery as well as routine analysis of blood biomarkers.

  • 17. Björling, Erik
    et al.
    Lindskog, Cecilia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Oksvold, Per
    Linné, Jerker
    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.
    Hober, Sophia
    Uhlén, Mathias
    Pontén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    A web-based tool for in silico biomarker discovery based on tissue-specific protein profiles in normal and cancer tissues2008In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 7, no 5, p. 825-844Article in journal (Refereed)
    Abstract [en]

    Here we report the development of a publicly available Web-based analysis tool for exploring proteins expressed in a tissue- or cancer-specific manner. The search queries are based on the human tissue profiles in normal and cancer cells in the Human Protein Atlas portal and rely on the individual annotation performed by pathologists of images representing immunohistochemically stained tissue sections. Approximately 1.8 million images representing more than 3000 antibodies directed toward human proteins were used in the study. The search tool allows for the systematic exploration of the protein atlas to discover potential protein biomarkers. Such biomarkers include tissue-specific markers, cell type-specific markers, tumor type-specific markers, markers of malignancy, and prognostic or predictive markers of cancers. Here we show examples of database queries to generate sets of candidate biomarker proteins for several of these different categories. Expression profiles of candidate proteins can then subsequently be validated by examination of the underlying high resolution images. The present study shows examples of search strategies revealing several potential protein biomarkers, including proteins specifically expressed in normal cells and in cancer cells from specified tumor types. The lists of candidate proteins can be used as a starting point for further validation in larger patient cohorts using both immunological approaches and technologies utilizing more classical proteomics tools.

  • 18. 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.

  • 19.
    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)
  • 20. Chornoguz, Olesya
    et al.
    Grmai, Lydia
    Sinha, Pratima
    Artemenko, Konstantin A.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Zubarev, Roman A.
    Ostrand-Rosenberg, Suzanne
    Proteomic Pathway Analysis Reveals Inflammation Increases Myeloid-Derived Suppressor Cell Resistance to Apoptosis2011In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 10, no 3Article in journal (Refereed)
    Abstract [en]

    Myeloid-derived suppressor cells (MDSC) accumulate in patients and animals with cancer where they mediate systemic immune suppression and obstruct immune-based cancer therapies. We have previously demonstrated that inflammation, which frequently accompanies tumor onset and progression, increases the rate of accumulation and the suppressive potency of MDSC. To determine how inflammation enhances MDSC levels and activity we used mass spectrometry to identify proteins produced by MDSC induced in highly inflammatory settings. Proteomic pathway analysis identified the Fas pathway and caspase network proteins, leading us to hypothesize that inflammation enhances MDSC accumulation by increasing MDSC resistance to Fas-mediated apoptosis. The MS findings were validated and extended by biological studies. Using activated caspase 3 and caspase 8 as indicators of apoptosis, flow cytometry, confocal microscopy, and Western blot analyses demonstrated that inflammation-induced MDSC treated with a Fas agonist contain lower levels of activated caspases, suggesting that inflammation enhances resistance to Fas-mediated apoptosis. Resistance to Fas-mediated apoptosis was confirmed by viability studies of MDSC treated with a Fas agonist. These results suggest that an inflammatory environment, which is frequently present in tumor-bearing individuals, protects MDSC against extrinsic-induced apoptosis resulting in MDSC with a longer in vivo half-life, and may explain why MDSC accumulate more rapidly and to higher levels in inflammatory settings.

  • 21. Clark, Gary F.
    et al.
    Grassi, Paola
    Pang, Poh-Choo
    Panico, Maria
    Lafrenz, David
    Drobnis, Erma Z.
    Baldwin, Michael R.
    Morris, Howard R.
    Haslam, Stuart M.
    Schedin-Weiss, Sophia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Sun, Wei
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Dell, Anne
    Tumor Biomarker Glycoproteins in the Seminal Plasma of Healthy Human Males Are Endogenous Ligands for DC-SIGN2012In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 11, no 1Article in journal (Refereed)
    Abstract [en]

    DC-SIGN is an immune C-type lectin that is expressed on both immature and mature dendritic cells associated with peripheral and lymphoid tissues in humans. It is a pattern recognition receptor that binds to several pathogens including HIV-1, Ebola virus, Mycobacterium tuberculosis, Candida albicans, Helicobacter pylori, and Schistosoma mansoni. Evidence is now mounting that DC-SIGN also recognizes endogenous glycoproteins, and that such interactions play a major role in maintaining immune homeostasis in humans and mice. Autoantigens (neoantigens) are produced for the first time in the human testes and other organs of the male urogenital tract under androgenic stimulus during puberty. Such antigens trigger autoimmune orchitis if the immune response is not tightly regulated within this system. Endogenous ligands for DC-SIGN could play a role in modulating such responses. Human seminal plasma glycoproteins express a high level of terminal Lewis(x) and Lewis(y) carbohydrate antigens. These epitopes react specifically with the lectin domains of DC-SIGN. However, because the expression of these sequences is necessary but not sufficient for interaction with DC-SIGN, this study was undertaken to determine if any seminal plasma glycoproteins are also endogenous ligands for DC-SIGN. Glycoproteins bearing terminal Lewis(x) and Lewis(y) sequences were initially isolated by lectin affinity chromatography. Protein sequencing established that three tumor biomarker glycoproteins (clusterin, galectin-3 binding glycoprotein, prostatic acid phosphatase) and protein C inhibitor were purified by using this affinity method. The binding of DC-SIGN to these seminal plasma glycoproteins was demonstrated in both Western blot and immunoprecipitation studies. These findings have confirmed that human seminal plasma contains endogenous glycoprotein ligands for DC-SIGN that could play a role in maintaining immune homeostasis both in the male urogenital tract and the vagina after coitus.

  • 22. Cornett, Dale S
    et al.
    Mobley, James A
    Dias, Eduardo C
    Andersson, Malin
    Vanderbilt University.
    Arteaga, Carlos L
    Sanders, Melinda E
    Caprioli, Richard M
    A novel histology-directed strategy for MALDI-MS tissue profiling that improves throughput and cellular specificity in human breast cancer.2006In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 5, no 10, p. 1975-83Article in journal (Refereed)
    Abstract [en]

    We describe a novel tissue profiling strategy that improves the cellular specificity and analysis throughput of protein profiles obtained by direct MALDI analysis. The new approach integrates the cellular specificity of histology, the accuracy and reproducibility of robotic liquid dispensing, and the speed and objectivity of automated spectra acquisition. Traditional methodologies for preparing and analyzing tissue samples rely heavily on manual procedures, which for various reasons discussed, restrict cellular specificity and sample throughput. Here, a robotic spotter deposits micron-sized droplets of matrix precisely onto foci of normal mammary epithelium, ductal carcinoma in situ, invasive mammary cancer, and peritumoral stroma selected by a pathologist from high resolution histological images of sectioned human breast cancer samples. The location of each matrix spot was then determined and uploaded into the instrument to facilitate automated profile acquisition by MALDI-TOF. In the example shown, the different lesions were clearly differentiated using mass profiling. Further, the workflow permits a visual projection of any information produced from the profile analyses directly on the histological image for a unique combination of proteomic and histological assessment of sample regions. The higher performance characteristics offered by the new workflow promises to be a significant advancement toward the next generation of tissue profiling studies.

  • 23.
    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)
  • 24.
    Darmanis, Spyros
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Nong, Rachel Yuan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Hammond, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Gu, Jijuan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Alderborn, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Vänelid, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Siegbahn, Agneta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Gustafsdottir, Sigrun
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Ericsson, Olle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Sensitive plasma protein analysis by microparticle-based proximity ligation assays2010In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 9, no 2, p. 327-335Article in journal (Refereed)
    Abstract [en]

    Detection of proteins released in the bloodstream from tissues damaged by disease can promote early detection of pathological conditions, differential diagnostics, and follow-up of therapy. Despite these prospects and a plethora of candidate biomarkers, efforts in recent years to establish new protein diagnostic assays have met with limited success. One important limiting factor has been the challenge of detecting proteins present at trace levels in complex bodily fluids. To achieve robust, sensitive, and specific detection, we have developed a microparticle-based solid-phase proximity ligation assay, dependent on simultaneous recognition of target proteins by three antibody molecules for added specificity. After capture on a microparticle, solid-phase pairs of proximity probes are added followed by washes, enabling detection and identification of rare protein molecules in blood while consuming small amounts of sample. We demonstrate that single polyclonal antibody preparations raised against target proteins of interest can be readily used to establish assays where detection depends on target recognition by three individual antibody molecules, recognizing separate epitopes. The assay was compared with state-of-the-art sandwich ELISAs for detection of vascular endothelial growth factor, interleukin-8 and interleukin-6, and it was found to be superior both with regard to dynamic range and minimal numbers of molecules detected. Furthermore, the assays exhibited excellent performance in undiluted plasma and serum as well as in whole blood, producing comparable results for nine different antigens. We thus show that solid-phase proximity ligation assay is suitable for validation of a variety of protein biomarkers over broad dynamic ranges in clinical samples.

  • 25. 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.

  • 26.
    Dubois, Louise
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Biochemial structure and function.
    Ronquist, K Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Biochemial structure and function.
    Ek, Bo
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Ronquist, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Biochemial structure and function.
    Larsson, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Biochemial structure and function.
    Proteomic profiling of detergent resistant membranes (lipid rafts) of prostasomes2015In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 14, no 11, p. 3015-3022Article in journal (Refereed)
    Abstract [en]

    Prostasomes are exosomes derived from prostate epithelial cells through exocytosis by multivesicular bodies. Prostasomes have a bilayered membrane and readily interact with sperm. The membrane lipid composition is unusual with a high contribution of sphingomyelin at the expense of phosphatidylcholine and saturated and monounsaturated fatty acids are dominant. Lipid rafts are liquid-ordered domains that are more tightly packed than the surrounding non-raft phase of the bilayer. Lipid rafts are proposed to be highly dynamic, submicroscopic assemblies that float freely within the liquid disordered membrane bilayer and some proteins preferentially partition into the ordered raft domains. We asked the question whether lipid rafts do exist in prostasomes and, if so, which proteins might be associated with them. Prostasomes of density range 1.13-1.19g/mL were subjected to density gradient ultracentrifugation in sucrose fabricated by phosphate buffered saline (PBS) containing 1% Triton X-100 with capacity for banding at 1.10g/mL, i.e. the classical density of lipid rafts. Prepared prostasomal lipid rafts (by gradient ultracentrifugation) were analyzed by mass spectrometry and electron microscopy. The clearly visible band on top of 1.10g/mL sucrose in the Triton X-100 containing gradient was subjected to LC-MS/MS and more than 370 lipid raft associated proteins were identified. Several of them were involved in intraluminal vesicle formation, e.g. tetraspanins, ESCRTs and Ras-related proteins. This is the first comprehensive LC-MS/MS profiling of proteins in lipid rafts derived from exosomes. Data are available via ProteomeXchange with identifier PXD002163.

  • 27.
    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.

  • 28. 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.

  • 29.
    El-Rami, Fadi E.
    et al.
    Pharmaceutical Sciences, Oregon State University, United States of America.
    Zielke, Ryszard A.
    College of Pharmacy, Oregon State University, United States of America.
    Wi, Teodora
    World Health Organization, Geneva, Switzerland.
    Sikora, Aleksandra E.
    Department of Pharmaceutical Sciences, Oregon State University, United States of America; Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton OR, United States.
    Unemo, Magnus
    Örebro University, School of Medical Sciences. Örebro University Hospital. World Health Organization, Collaborating Centre for Gonorrhoea and Other Sexually Transmitted Infections, Department of Laboratory Medicine, Clinical Microbiology.
    Quantitative proteomics of the 2016 WHO Neisseria gonorrhoeae reference strains surveys vaccine candidates and antimicrobial resistance determinants2019In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 18, no 1, p. 127-150Article in journal (Refereed)
    Abstract [en]

    The sexually transmitted disease gonorrhea (causative agent: Neisseria gonorrhoeae) remains an urgent public health threat globally due to its reproductive health repercussions, high incidence, widespread antimicrobial resistance (AMR), and absence of a vaccine. To mine gonorrhea antigens and enhance our understanding of gonococcal AMR at the proteome level, we performed the first large-scale proteomic profiling of a diverse panel (n=15) of gonococcal strains, including the 2016 World Health Organization (WHO) reference strains. These strains show all existing AMR profiles - established through phenotypic characterization and reference genome publication - and are intended for quality assurance in laboratory investigations. Herein, these isolates were subjected to subcellular fractionation and labeling with tandem mass tags coupled to mass spectrometry and multi-combinatorial bioinformatics. Our analyses detected 904 and 723 common proteins in cell envelope and cytoplasmic subproteomes, respectively. We identified nine novel gonorrhea vaccine candidates. Expression and conservation of new and previously selected antigens were investigated. In addition, established gonococcal AMR determinants were evaluated for the first time using quantitative proteomics. Six new proteins, WHO_F_00238, WHO_F_00635c, WHO_F_00745, WHO_F_01139, WHO_F_01144c, and WHO_F_01126, were differentially expressed in all strains, suggesting that they represent global proteomic AMR markers, indicate a predisposition toward developing or compensating gonococcal AMR, and/or act as new antimicrobial targets. Finally, phenotypic clustering based on the isolates' defined antibiograms and common differentially expressed proteins yielded seven matching clusters between established and proteome-derived AMR signatures. Together, our investigations provide a reference proteomics databank for gonococcal vaccine and AMR research endeavors, which enables microbiological, clinical, or epidemiological projects and enhances the utility of the WHO reference strains.

  • 30.
    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)
  • 31.
    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.

  • 32. Fagerberg, Linn
    et al.
    Hallström, Björn M
    Oksvold, Per
    Kampf, Caroline
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Djureinovic, Dijana
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Odeberg, Jacob
    Habuka, Masato
    Tahmasebpoor, Simin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Danielsson, Angelika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Edlund, Karolina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Asplund, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Sjöstedt, Evelina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Lundberg, Emma
    Szigyarto, Cristina Al-Khalili
    Skogs, Marie
    Takanen, Jenny Ottosson
    Berling, Holger
    Tegel, Hanna
    Mulder, Jan
    Nilsson, Peter
    Schwenk, Jochen M
    Lindskog, Cecilia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Danielsson, Frida
    Mardinoglu, Adil
    Sivertsson, Asa
    von Feilitzen, Kalle
    Forsberg, Mattias
    Zwahlen, Martin
    Olsson, IngMarie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Navani, Sanjay
    Huss, Mikael
    Nielsen, Jens
    Pontén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Uhlén, Mathias
    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.

  • 33.
    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)
  • 34.
    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.

  • 35.
    Fälth, Maria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Sköld, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Norrman, Mathias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Svensson, Marcus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Fenyö, David
    Andrén, Per E
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    SwePep – A database designed for endogenous peptides and mass spectrometry2006In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 5, no 6, p. 998-1005Article in journal (Refereed)
    Abstract [en]

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

  • 36.
    Fälth, Maria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Sköld, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Norrman, Mathias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Svensson, Marcus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Fenyö, David
    Andrén, Per E
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    SwePep, a database designed for endogenous peptides and mass spectrometry2006In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 5, no 6, p. 998-1005Article in journal (Refereed)
    Abstract [en]

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

  • 37.
    Fälth, Maria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Sköld, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Svensson, Marcus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Nilsson, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Fenyö, David
    Andrén, Per E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
    Neuropeptidomics strategies for specific and sensitive identification of endogenous peptides2007In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 6, no 7, p. 1188-1197Article in journal (Refereed)
    Abstract [en]

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

  • 38. 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.

  • 39. Geiger, Tamar
    et al.
    Velic, Ana
    Macek, Boris
    Lundberg, Emma
    Kampf, Caroline
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Nagaraj, Nagarjuna
    Uhlen, Mathias
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Cox, Juergen
    Mann, Matthias
    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.

  • 40.
    Gerold, Gisa
    et al.
    Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover and the Helmholtz Centre for Infection Research, Hannover, Germany.
    Bruening, Janina
    Weigel, Bettina
    Pietschmann, Thomas
    Protein Interactions during the Flavivirus and Hepacivirus Life Cycle2017In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 16, no 4, p. 75-91Article in journal (Refereed)
    Abstract [en]

    interaction proteomics and why we believe these challenges should be met.

  • 41. Gloriam, David E.
    et al.
    Orchard, Sandra
    Bertinetti, Daniela
    Björling, Erik
    Bongcam-Rudloff, Erik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    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
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Montecchi-Palazzi, Luisa
    Palcy, Sandrine
    Rodriguez, Henry
    Schweinsberg, Sonja
    Sievert, Volker
    Stoevesandt, Oda
    Taussig, Michael J.
    Ueffing, Marius
    Uhlén, Mathias
    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.

  • 42. 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.

  • 43.
    Götzke, Hansjörg
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Muheim, Claudio
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Altelaar, A. F. Maarten
    Heck, Albert J. R.
    Maddalo, Gianluca
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Utrecht University, The Netherlands; Netherlands Proteomics Centre, The Netherlands.
    Daley, Daniel O.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Identification of Putative Substrates for the Periplasmic Chaperone YfgM in Escherichia coli Using Quantitative Proteomics2015In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 14, no 1, p. 216-226Article in journal (Refereed)
    Abstract [en]

    How proteins are trafficked, folded, and assembled into functional units in the cell envelope of Gram-negative bacteria is of significant interest. A number of chaperones have been identified, however, the molecular roles of these chaperones are often enigmatic because it has been challenging to assign substrates. Recently we discovered a novel periplasmic chaperone, called YfgM, which associates with PpiD and the SecYEG translocon and operates in a network that contains Skp and SurA. The aim of the study presented here was to identify putative substrates of YfgM. We reasoned that substrates would be incorrectly folded or trafficked when YfgM was absent from the cell, and thus more prone to proteolysis (the loss-of-function rationale). We therefore used a comparative proteomic approach to identify cell envelope proteins that were lower in abundance in a strain lacking yfgM, and strains lacking yfgM together with either skp or surA. Sixteen putative substrates were identified. The list contained nine inner membrane proteins (CusS, EvgS, MalF, OsmC, TdcB, TdcC, WrbA, YfhB, and YtfH) and seven periplasmic proteins (HdeA, HdeB, AnsB, Ggt, MalE, YcgK, and YnjE), but it did not include any lipoproteins or outer membrane proteins. Significantly, AnsB (an asparaginase) and HdeB (a protein involved in the acid stress response), were lower in abundance in all three strains lacking yfgM. For both genes, we ruled out the possibility that they were transcriptionally down-regulated, so it is highly likely that the corresponding proteins are misfolded/mistargeted and turned-over in the absence of YfgM. For HdeB we validated this conclusion in a pulse-chase experiment. The identification of HdeB and other cell envelope proteins as potential substrates will be a valuable resource for follow-up experiments that aim to delineate molecular the function of YfgM.

  • 44.
    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.

  • 45.
    Hanrieder, Jörg
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Ljungdahl, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Fälth, Maria
    Eriksson Mammo, Sofie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Andersson, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    L-DOPA-induced dyskinesia is associated with regional increase of striatal dynorphin peptides as elucidated by imaging mass spectrometry2011In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 10, no 10, p. M111.009308-Article in journal (Refereed)
    Abstract [en]

    Opioid peptides are involved in various pathophysiological processes, including algesia, epilepsy and drug dependency. A strong association between L-DOPA-induced dyskinesia (LID) and elevated prodynorphin mRNA levels has been established in both patients and in animal models of Parkinsons disease (PD), but to date the endogenous prodynorphin peptide products have not been determined. Here, matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) was used for characterization, localization, and relative quantification of striatal neuropeptides in a rat model of LID in PD. MALDI-IMS has the unique advantage of high sensitivity and high molecular specificity, allowing comprehensive detection of multiple molecular species in a single tissue section. Indeed, several dynorphins and enkephalins could be detected in the present study, including dynorphin B, alpha-neoendorphin, MetEnkRF, MetEnkRGL, PEnk (198-209, 219-229). IMS analysis revealed elevated levels of dynorphin B, alpha-neoendorphin, substance P, and PEnk (220-229) in the dorsolateral striatum of high-dyskinetic animals compared to low-dyskinetic and lesion-only control rats. Here, only peak -intensities of the prodynorphin-derived peptides, dynorphin B and alpha-neoendorphin, were strongly and positively correlated with LID severity. Interestingly, these LID associated dynorphin peptides are not mainly those with high affinity to kappa opioid receptors, but are known to bind and activate also mu- and delta-opioid receptors. In addition, the peak intensities of a putative metabolite of alpha-neoendorphin lacking the N-terminal tyrosine correlated positively with dyskinesia severity. Des-tyrosine dynorphins display reduced opioid receptor binding and this points to possible compensatory non-opioid mediated changes in the striatum. Since des-tyrosine dynorphins can only be detected by mass spectrometry, as no antibodies are currently available, these findings highlight the potential of MALDI-IMS analysis for the study of molecular dynamics in neurological diseases. This is the first MALDI-IMS-based study on neuropeptide analysis in experimental PD and LID. This unique methodological approach facilitated comprehensive investigation of LID-associated prodynorphin-derived peptide products.

  • 46.
    Huang, F
    et al.
    Stockholm University.
    Parmryd, I
    Stockholm University.
    Nilsson, F
    AstraZeneca R&D .
    Persson, AL
    Stockholm University.
    Pakrasi, HB
    Washington University.
    Andersson, Bertil
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Norling, B
    Stockholm University.
    Proteomics of Synechocystis sp strain PCC 6803 - Identification of plasma membrane proteins2002In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 1, no 12, p. 956-966Article in journal (Refereed)
    Abstract [en]

    Cyanobacteria are unique prokaryotes since they in addition to outer and plasma membranes contain the photosynthetic membranes (thylakoids) The plasma membranes of Synechocystis 6803, which can be completely purified by density centrifugation and polymer two-phase partitioning, have been found to be more complex than previously anticipated, i. e they appear to be essential for assembly of the two photosystems. A proteomic approach for the characterization of cyanobacterial plasma membranes using two-dimensional gel electrophoresis and mass spectrometry analysis revealed a total of 57 different membrane proteins of which 17 are integral membrane spanning proteins. Among the 40 peripheral proteins 20 are located on the periplasmic side of the membrane, while 20 are on the cytoplasmic side. Among the proteins identified are subunits of the two photosystems as well as Vipp1, which has been suggested to be involved in vesicular transport between plasma and thylakoid membranes and is thus relevant to the possibility that plasma membranes are the initial site for photosystem biogenesis. Four subunits of the Pilus complex responsible for cell motility were also identified as well as several subunits of the TolC and TonB transport systems. Several periplasmic and ATP-binding proteins of ATP-binding cassette transporters were also identified as were two subunits of the F-0 membrane part of the ATP synthase

  • 47.
    Huang, Fang
    et al.
    Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-10691 Stockholm, Sweden.
    Parmryd, Ingela
    Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-10691 Stockholm, Sweden.
    Nilsson, Fredrik
    AstraZeneca R&D Mölndal, SE-43183 Mölndal, Sweden .
    Persson, Annika L.
    Department of Zoological Cell Biology, The Wenner-Gren Institute, Stockholm University, SE-10691 Stockholm, Sweden.
    Pakrasi, Himadri B.
    Department of Biology, Washington University, St. Louis, Missouri 63130.
    Andersson, Bertil
    Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-10691 Stockholm, Sweden;Division of Cell Biology, Linköping University, SE-58185 Linköping, Sweden.
    Norling, Birgitta
    Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-10691 Stockholm, Sweden.
    Proteomics of Synechocystis sp. strain PCC 6803: identification of plasma membrane proteins2002In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 1, no 12, p. 956-966Article in journal (Refereed)
    Abstract [en]

    Cyanobacteria are unique prokaryotes since they in addition to outer and plasma membranes contain the photosynthetic membranes (thylakoids). The plasma membranes of Synechocystis 6803, which can be completely purified by density centrifugation and polymer two-phase partitioning, have been found to be more complex than previously anticipated, i.e. they appear to be essential for assembly of the two photosystems. A proteomic approach for the characterization of cyanobacterial plasma membranes using two-dimensional gel electrophoresis and mass spectrometry analysis revealed a total of 57 different membrane proteins of which 17 are integral membrane spanning proteins. Among the 40 peripheral proteins 20 are located on the periplasmic side of the membrane, while 20 are on the cytoplasmic side. Among the proteins identified are subunits of the two photosystems as well as Vipp1, which has been suggested to be involved in vesicular transport between plasma and thylakoid membranes and is thus relevant to the possibility that plasma membranes are the initial site for photosystem biogenesis. Four subunits of the Pilus complex responsible for cell motility were also identified as well as several subunits of the TolC and TonB transport systems. Several periplasmic and ATP-binding proteins of ATP-binding cassette transporters were also identified as were two subunits of the F(0) membrane part of the ATP synthase.

  • 48.
    Häggmark, Anna
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics.
    Bradley, Frideborg
    Karolinska Univ Hosp, Ctr Mol Med, Karolinska Inst, Dept Med Solna,Unit Infect Dis, Stockholm, Sweden..
    Qundos, Ulrika
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics.
    Guthrie, Brandon L.
    Univ Washington, Dept Global Hlth, Washington, DC USA.;Univ Washington, Dept Epidemiol Hlth, Washington, DC USA..
    Birse, Kenzie
    Univ Manitoba, Dept Med Microbiol, Winnipeg, MB, Canada.;Publ Hlth Agcy Canada, JC Wilt Infect Dis Ctr, Natl HIV & Retrovirol Labs, Winnipeg, MB, Canada..
    Noel-Romas, Laura
    Univ Manitoba, Dept Med Microbiol, Winnipeg, MB, Canada.;Publ Hlth Agcy Canada, JC Wilt Infect Dis Ctr, Natl HIV & Retrovirol Labs, Winnipeg, MB, Canada..
    Lindskog, Cecilia
    Uppsala Univ, Dept Immunol Genet & Pathol, SciLifeLab, Uppsala, Sweden..
    Bosire, Rose
    Kenya Govt Med Res Ctr, Nairobi, Kenya..
    Kiarie, James
    Univ Nairobi, Dept Obstet & Gynecol, Nairobi, Kenya..
    Farquhar, Carey
    Univ Washington, Dept Med Global Hlth & Epidemiol, Seattle, WA 98195 USA..
    Burgener, Adam D.
    Karolinska Univ Hosp, Ctr Mol Med, Karolinska Inst, Dept Med Solna,Unit Infect Dis, Stockholm, Sweden.;Univ Manitoba, Dept Med Microbiol, Winnipeg, MB, Canada.;Publ Hlth Agcy Canada, JC Wilt Infect Dis Ctr, Natl HIV & Retrovirol Labs, Winnipeg, MB, Canada..
    Nilsson, Peter
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics.
    Broliden, Kristina
    Karolinska Univ Hosp, Ctr Mol Med, Karolinska Inst, Dept Med Solna,Unit Infect Dis, Stockholm, Sweden..
    A High-throughput Bead-based Affinity Assay Enables Analysis of Genital Protein Signatures in Women At Risk of HIV Infection2019In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 18, no 3, p. 461-476Article in journal (Refereed)
    Abstract [en]

    Women at high risk of HIV infection, including sex workers and those with active genital inflammation, have molecular signatures of immune activation and epithelial barrier remodeling in samples of their genital mucosa. These alterations in the local immunological milieu are likely to impact HIV susceptibility. We here analyze host genital protein signatures in HIV uninfected women, with high frequency of condom use, living in HIV-serodiscordant relationships. Cervicovaginal secretions from women living in HIV-serodiscordant relationships (n = 62) were collected at three time points over 12 months. Women living in HIV-negative seroconcordant relationships (controls, n = 25) were sampled at one time point. All study subjects were examined for demographic parameters associated with susceptibility to HIV infection. The cervicovaginal samples were analyzed using a high-throughput bead-based affinity assay. Proteins involved in epithelial barrier function and inflammation were increased in HIV-serodiscordant women. By combining several methods of analysis, a total of five proteins (CAPG, KLK10, SPRR3, elafin/PI3, CSTB) were consistently associated with this study group. Proteins analyzed using the affinity set-up were further validated by label-free tandem mass spectrometry in a partially overlapping cohort with concordant results. Women living in HIV-serodiscordant relationships thus had elevated levels of proteins involved in epithelial barrier function and inflammation despite low prevalence of sexually transmitted infections and a high frequency of safe sex practices. The identified proteins are important markers to follow during assessment of mucosal HIV susceptibility factors and a high-throughput bead-based affinity set-up could be a suitable method for such evaluation.

  • 49. 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.

  • 50. Janzi, M.
    et al.
    Ödling, Jenny
    KTH, Molekylär Bioteknologi.
    Pan-Hammarstrom, Q.
    Sundberg, Mårten
    KTH, Proteomik.
    Lundeberg, Joakim
    KTH, Genteknologi.
    Uhlén, Mathias
    KTH, Proteomik.
    Hammarstrom, L.
    Nilsson, Peter
    KTH, Proteomik.
    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.

123 1 - 50 of 127
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