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  • 1. Colwill, Karen
    et al.
    Nilsson, Peter
    KTH, Proteomik.
    Sundberg, Mårten
    KTH, Proteomik.
    Sjöberg, Ronald
    KTH, Proteomik.
    Sivertsson, Åsa
    KTH, Proteomik.
    Schwenk, Jochen M
    KTH, Proteomik.
    Ottosson Takanen, Jenny
    KTH, Proteomik.
    Hober, Sophia
    KTH, Proteomik.
    Uhlén, Mathias
    KTH, Proteomik.
    Gräslund, Susanne
    A roadmap to generate renewable protein binders to the human proteome2011In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 8, no 7, p. 551-8Article in journal (Refereed)
    Abstract [en]

    Despite the wealth of commercially available antibodies to human proteins, research is often hindered by their inconsistent validation, their poor performance and the inadequate coverage of the proteome. These issues could be addressed by systematic, genome-wide efforts to generate and validate renewable protein binders. We report a multicenter study to assess the potential of hybridoma and phage-display technologies in a coordinated large-scale antibody generation and validation effort. We produced over 1,000 antibodies targeting 20 SH2 domain proteins and evaluated them for potency and specificity by enzyme-linked immunosorbent assay (ELISA), protein microarray and surface plasmon resonance (SPR). We also tested selected antibodies in immunoprecipitation, immunoblotting and immunofluorescence assays. Our results show that high-affinity, high-specificity renewable antibodies generated by different technologies can be produced quickly and efficiently. We believe that this work serves as a foundation and template for future larger-scale studies to create renewable protein binders.

  • 2.
    Eriksson, Cecilia
    et al.
    KTH, Proteomik.
    Agaton, Charlotta
    KTH, Skolan för bioteknologi (BIO).
    Kånge, Rikard
    Sundberg, Marten
    KTH, Skolan för bioteknologi (BIO).
    Nilsson, Peter
    KTH, Skolan för bioteknologi (BIO).
    Ek, Bo
    Uhlen, Mathias
    KTH, Proteomik.
    Gustafsson, Magnus
    Hober, Sophia
    KTH, Proteomik.
    Microfluidic analysis of antibody specificity in a compact disk format2006In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 5, no 7, p. 1568-1574Article in journal (Refereed)
    Abstract [en]

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

  • 3. 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.

  • 4.
    Lundberg, Emma
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Sundberg, Mårten
    KTH, Skolan för bioteknologi (BIO).
    Gräslund, Torbjörn
    KTH, Skolan för bioteknologi (BIO).
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO).
    Andersson-Svahn, Helene
    KTH, Skolan för bioteknologi (BIO).
    A novel method for reproducible fluorescent labeling of small amounts of antibodies on solid phase2007In: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 322, no 1-2, p. 40-49Article in journal (Refereed)
    Abstract [en]

    Fluorescently labeled antibodies are very important tools in cell biology, providing for specific and quantitative detection of antigens. To date, fluorophore labeling of antibodies has been performed in solution and has been limited by low-throughput methods requiring a substantial amount of pure antibody sample at a high concentration. We have developed a novel solid-phase labeling protocol for small amounts (i.e. micrograms) of antibodies with fluorescent dyes. Protein A affinity medium was used as solid support in a micropipette tip format. This solid-phase approach, including the advantage of the strong and specific interaction between Protein A and antibodies, allows for simultaneous purification, labeling and concentration of the antibody sample, making it possible to start with unpure antibody samples at low concentrations. We have optimized the protocol with regard to reaction pH, time, temperature and amount of amine reactive dye. In addition, we have evaluated the stability and activity of the labeled antibodies. To evaluate the reproducibility and robustness of this method we labeled eight antibodies with amine reactive fluorescent dyes followed by evaluation of antibody specificity on protein arrays. Interestingly, this gave an extremely high conformity in the degree of labeling, showing the robustness of the method. The solid-phase method also gave predictable and reproducible results and by varying the amount of reactive dye, the desired degree of labeling can easily be achieved. Antibodies labeled using this solid-phase method were similar in stability and activity to antibodies labeled in solution. This novel solid-phase antibody labeling method may also be applicable for other conjugation chemistries and labels, and has potential for throughput applications.

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

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

  • 6.
    Rimini, Rebecca
    et al.
    KTH, Proteomik.
    Schwenk, Jochen M.
    KTH, Proteomik.
    Sundberg, Marten
    Sjöberg, Ronald
    Klevebring, Daniel
    Gry, Marcus
    KTH, Proteomik.
    Uhlén, Mathias
    KTH, Proteomik.
    Nilsson, Peter
    KTH, Proteomik.
    Validation of serum protein profiles by a dual antibody array approach2009In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 73, no 2, p. 252-266Article in journal (Refereed)
    Abstract [en]

    In recent years, affinity-based technologies have become important tools for serum profiling to uncover protein expression patterns linked to disease state or therapeutic effects. In this study, we describe a path towards the production of an antibody microarray to allow protein profiling of biotinylated human serum samples with reproducible sensitivity in the picomolar range. With the availability of growing numbers of affinity reagents, protein profiles are to be validated in efficient manners and we describe a cross-platform strategy based on data concordance with a suspension bead array to interrogate the identical set of antibodies with the same cohort of serum samples. Comparative analysis enabled to screen for high-performing antibodies, which were displaying consistent results across the two platforms and targeting known serum components. Moreover, data processing methods such as sample referencing and normalization were evaluated for their effects on inter-platform agreement. Our work suggests that mutual validation of protein expression profiles using alternative microarray platforms holds great potential in becoming an important and valuable component in affinity-based high-throughput proteomic screenings as it allows to narrow down the number of discovered targets prior to orthogonal, uniplexed validation approaches.

  • 7.
    Schwenk, Jochen M.
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Lindberg, Johan
    Sundberg, Mårten
    KTH, Proteomik.
    Uhlén, Mathias
    KTH, Proteomik.
    Nilsson, Peter
    KTH, Proteomik.
    Determination of binding specificities in highly multiplexed bead-based assays for antibody proteomics2007In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 6, no 1, p. 125-132Article in journal (Refereed)
    Abstract [en]

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

  • 8.
    Sjöberg, Ronald
    et al.
    KTH, Proteomik.
    Sundberg, Mårten
    KTH, Proteomik.
    Gundberg, Anna
    KTH, Skolan för bioteknologi (BIO).
    Sivertsson, Åsa
    KTH, Skolan för bioteknologi (BIO).
    Schwenk, Jochen M.
    KTH, Skolan för bioteknologi (BIO).
    Uhlén, Mathias
    KTH, Proteomik.
    Nilsson, Peter
    KTH, Proteomik.
    Validation of affinity reagents using antigen microarrays2011In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 29, no 5, p. 555-563Article in journal (Other academic)
    Abstract [en]

    There is a need for standardised validation of affinity reagents to determine their binding selectivity and specificity. This is of particular importance for systematic efforts that aim to cover the human proteome with different types of binding reagents. One such international program is the SH2-consortium, which was formed to generate a complete set of renewable affinity reagents to the SH2-domain containing human proteins. Here, we describe a microarray strategy to validate various affinity reagents, such as recombinant single-chain antibodies, mouse monoclonal antibodies and antigen-purified polyclonal antibodies using a highly multiplexed approach. An SH2-specific antigen microarray was designed and generated, containing more than 6000 spots displayed by 14 identical subarrays each with 406 antigens, where 105 of them represented SH2-domain containing proteins. Approximately 400 different affinity reagents of various types were analysed on these antigen microarrays carrying antigens of different types. The microarrays revealed not only very detailed specificity profiles for all the binders, but also showed that overlapping target sequences of spotted antigens were detected by off-target interactions. The presented study illustrates the feasibility of using antigen microarrays for integrative, high-throughput validation of various types of binders and antigens.

  • 9. Strage, Emma M
    et al.
    Sundberg, Mårten
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Holst, Bodil S
    Andersson Franko, Mikael
    Ramström, Margareta
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Fall, Tove
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Molecular epidemiology.
    Lewitt, Moira
    Effect of insulin treatment on circulating insulin-like growth factor I and IGF-binding proteins in cats with diabetes mellitus.2018In: Journal of Veterinary Internal Medicine, ISSN 0891-6640, E-ISSN 1939-1676, Vol. 32, no 5, p. 1579-1590Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Insulin-like growth factor-I (IGF-I) is used to screen for acromegaly in diabetic cats. In humans, most circulating IGF-I forms ternary complexes (TC) with IGF-binding protein (IGFBP-3) and an acid-labile subunit. Compared to humans, the amount of TC in cats is more variable. Insulin-like growth factor-I concentrations are reported to increase during insulin treatment, more rapidly in cats achieving remission.

    OBJECTIVES: To investigate (i) factors associated with circulating IGF-I concentrations, including IGFBP-profiles (ii) effect of insulin treatment on IGF-I concentrations and (iii) IGF-I as prognostic marker of diabetes mellitus remission.

    ANIMALS: Thirty-one privately owned diabetic cats of which 24 were followed 1 year, and 13 healthy cats.

    METHODS: Prospective study. Serum insulin, IGF-I, glucose, and fructosamine concentrations were measured. IGF-binding forms were determined by chromatography in 14 diabetic and 13 healthy cats; and IGF-I, IGF-II, IGFBP-3, and IGFBP-5 by mass spectrometry in 3 cats achieving remission.

    RESULTS: Insulin-like growth factor-I median (interquartile range) before start of insulin treatment was 300 (160-556) ng/mL. Insulin-like growth factor-I was positively associated with TC (P < .0001) and endogenous insulin (P = .005) and negatively associated with fructosamine (P < .0001). Median IGF-I was higher 2-4 weeks after start of insulin treatment compared with baseline (300 versus 670 ng/mL, P = .0001) and predicted future remission (P = .046). In cats that went into remission, the amount of TC and IGFBP-3 increased, suggesting increase in IGF-I is dependent on TC formation.

    CONCLUSIONS: Insulin treatment should be accounted for when interpreting IGF-I in diabetic cats. Insulin-like growth factor-I 2-4 weeks after initiation of insulin treatment shows promise as prognostic marker for remission in diabetic cats.

  • 10.
    Sundberg, Mårten
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Mass Spectrometry and Affinity Based Methods for Analysis of Proteins and Proteomes2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Proteomics is a fast growing field and there has been a tremendous increase of knowledge the last two decades. Mass spectrometry is the most used method for analysis of complex protein samples. It can be used both in large scale discovery studies as well as in targeted quantitative studies. In parallel with the fast improvements of mass spectrometry-based proteomics there has been a fast growth of affinity-based methods. A common challenge is the large dynamic range of protein concentrations in biological samples. No method can today cover the whole dynamic range. If affinity and mass spectrometry-based proteomics could be used in better combination, this would be partly solved. The challenge for affinity-based proteomics is the poor specificity that has been seen for many of the commercially available antibodies. In mass spectrometry, the challenges are sensitivity and sample throughput. In this thesis, large scale approaches for validation of antibodies and other binders are presented. Protein microarrays were used in four validation studies and one was based on mass spectrometry. It is shown that protein microarrays can be valuable tools to check the specificity of antibodies produced in a large scale production. Mass spectrometry was shown to give similar results as Western blot and Immunohistochemistry regarding specificity, but did also provide useful information about which other proteins that were bound to the antibody.

    Mass spectrometry has many applications and in this thesis two methods contributing with new knowledge in animal proteomics are presented. A combination of high affinity depletion, SDS PAGE and mass spectrometry revealed 983 proteins in dog cerebrospinal fluid, of which 801 were marked as uncharacterized in UniProt. A targeted quantitative study of cat serum based on parallel reaction monitoring showed that mass spectrometry can be an applicable method instead of ELISA in animal proteomic studies. Mass spectrometry is a generic method and has the advantage of shorter and less expensive development costs for specific assays that are not hampered by cross-reactivity.

    Mass spectrometry supported by affinity based applications will be an attractive tool for further improvements in the proteomic field.

    List of papers
    1. Towards a human proteome atlas: high-throughput generation of mono-specific antibodies for tissue profiling
    Open this publication in new window or tab >>Towards a human proteome atlas: high-throughput generation of mono-specific antibodies for tissue profiling
    Show others...
    2005 (English)In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 5, no 17, p. 4327-4337Article in journal (Refereed) Published
    Abstract [en]

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

    Keywords
    Antibody generation, protein microarray, proteome atlas, tissue microarray, tissue profiling
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-80602 (URN)10.1002/pmic.200500072 (DOI)16237735 (PubMedID)
    Note

    LP, KL och JÖ contributed equally to this work.

    Available from: 2007-04-18 Created: 2007-04-18 Last updated: 2018-06-04Bibliographically approved
    2. Microfluidic analysis of antibody specificity in a compact disk format
    Open this publication in new window or tab >>Microfluidic analysis of antibody specificity in a compact disk format
    Show others...
    2006 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 5, no 7, p. 1568-1574Article in journal (Refereed) Published
    Abstract [en]

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

    Keywords
    microfluidics, miniaturization, compact disk, biochip, gyrolab, antibody specificity, IMMUNOSORBENT-ASSAY ELISA, PROTEOMICS, MICROARRAYS, GENOME, CHIP
    National Category
    Industrial Biotechnology
    Identifiers
    urn:nbn:se:uu:diva-208954 (URN)10.1021/pr050447c (DOI)000238838500007 ()
    Note

    QC 20100712

    Available from: 2010-07-12 Created: 2013-10-11 Last updated: 2018-06-05
    3. Determination of binding specificities in highly multiplexed bead-based assays for antibody proteomics
    Open this publication in new window or tab >>Determination of binding specificities in highly multiplexed bead-based assays for antibody proteomics
    Show others...
    2007 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 6, no 1, p. 125-132Article in journal (Refereed) Published
    Abstract [en]

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

    Keywords
    protein microarrays, atlas
    National Category
    Industrial Biotechnology
    Identifiers
    urn:nbn:se:uu:diva-208952 (URN)10.1074/mcp.T60035-MCP200 (DOI)000243312000011 ()
    Note

    QC 20100525

    Available from: 2010-08-05 Created: 2013-10-11 Last updated: 2018-06-05
    4. Validation of affinity reagents using antigen microarrays
    Open this publication in new window or tab >>Validation of affinity reagents using antigen microarrays
    Show others...
    2011 (English)In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 29, no 5, p. 555-563Article in journal (Other academic) Published
    Abstract [en]

    There is a need for standardised validation of affinity reagents to determine their binding selectivity and specificity. This is of particular importance for systematic efforts that aim to cover the human proteome with different types of binding reagents. One such international program is the SH2-consortium, which was formed to generate a complete set of renewable affinity reagents to the SH2-domain containing human proteins. Here, we describe a microarray strategy to validate various affinity reagents, such as recombinant single-chain antibodies, mouse monoclonal antibodies and antigen-purified polyclonal antibodies using a highly multiplexed approach. An SH2-specific antigen microarray was designed and generated, containing more than 6000 spots displayed by 14 identical subarrays each with 406 antigens, where 105 of them represented SH2-domain containing proteins. Approximately 400 different affinity reagents of various types were analysed on these antigen microarrays carrying antigens of different types. The microarrays revealed not only very detailed specificity profiles for all the binders, but also showed that overlapping target sequences of spotted antigens were detected by off-target interactions. The presented study illustrates the feasibility of using antigen microarrays for integrative, high-throughput validation of various types of binders and antigens.

    Keywords
    protein microarray, antibody validation, affinity reagent, antigen, specificity, SH2
    National Category
    Industrial Biotechnology
    Identifiers
    urn:nbn:se:uu:diva-208949 (URN)10.1016/j.nbt.2011.11.009 (DOI)000305606500007 ()
    Available from: 2011-11-17 Created: 2013-10-11 Last updated: 2018-06-05
    5. Estrogen receptor beta profiling in human tissues following extensive antibody validation
    Open this publication in new window or tab >>Estrogen receptor beta profiling in human tissues following extensive antibody validation
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Keywords
    Estrogen receptor beta, ER-beta, IHC, antibody validation, TMA
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-251340 (URN)
    Available from: 2015-04-21 Created: 2015-04-15 Last updated: 2015-10-01
    6. Quantitative and selective analysis of feline growth related proteins using parallel reaction monitoring high resolution mass spectrometry
    Open this publication in new window or tab >>Quantitative and selective analysis of feline growth related proteins using parallel reaction monitoring high resolution mass spectrometry
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Keywords
    Parallel reaction monitoring, QPrEST, targeted proteomics, cat, feline, Orbitrap, mass spectrometry, IGF-I, IGF-II, IGFBP-3, IGFBP-5
    National Category
    Analytical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-259614 (URN)
    Available from: 2015-08-10 Created: 2015-08-10 Last updated: 2015-10-01
    7. High-abundant protein depletion strategies applied on dog cerebrospinal fluid and evaluated by high-resolution mass spectrometry
    Open this publication in new window or tab >>High-abundant protein depletion strategies applied on dog cerebrospinal fluid and evaluated by high-resolution mass spectrometry
    2015 (English)In: Biochemistry and Biophysics Reports, ISSN 2405-5808, Vol. 3, p. 68-75Article in journal (Refereed) Published
    Abstract [en]

    As the number of fully sequenced animal genomes and the performance of advanced mass spectrometry-based proteomics techniques are continuously improving, there is now a great opportunity to increase the knowledge of various animal proteomes. This research area is further stimulated by a growing interest from veterinary medicine and the pharmaceutical industry. Cerebrospinal fluid (CSF) is a good source for better understanding of diseases related to the central nervous system, both in humans and other animals. In this study, four high-abundant protein depletion columns, developed for human or rat serum, were evaluated for dog CSF. For the analysis, a shotgun proteomics approach, based on nanoLC-LTQ Orbitrap MS/MS, was applied. All the selected approaches were shown to deplete dog CSF with different success. It was demonstrated that the columns significantly improved the coverage of the detected dog CSF proteome. An antibody-based column showed the best performance, in terms of efficiency, repeatability and the number of proteins detected in the sample. In total 983 proteins were detected. Of those, 801 proteins were stated as uncharacterized in the UniProt database. To the best of our knowledge, this is the so far largest number of proteins reported for dog CSF in one single study.

    Keywords
    Cerebrospinal fluid, Dog, High-abundant protein depletion, Shotgun proteomics, Orbitrap, Mass spectrometry
    National Category
    Analytical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-259611 (URN)10.1016/j.bbrep.2015.07.013 (DOI)
    Available from: 2015-08-10 Created: 2015-08-10 Last updated: 2015-10-01Bibliographically approved
  • 11.
    Sundberg, Mårten
    KTH, Proteomik.
    Protein microarrays for validation of affinity binders2011Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    Is specificity an important issue regarding affinity reagents? What about the validation of affinity reagents today, is it good enough? This depends on the application and the producer of the reagent. Validation should be the most important marketing argument that can be found.Today there is a continuous growth of both the number of affinity reagents that are produced and the different types of affinity reagents that are developed. In proteomics they become more and more important in exploring the human proteome. Therefore, validated affinity reagents should be on top of every proteomic researcher’s list. How should this be accomplished?Better international agreements on how affinity reagents should be tested to be regarded as functional reagents are needed. One of the most important issues is the specificity of the affinity reagent. An international standard for which specific validation that is needed for different kinds of applications would be very useful.In this thesis, it is shown that the protein microarray platform that was established within the HPA project at KTH is a very good tool to determine the specificity of different affinity binders.In the first study, the production of mono-specific antibodies for tissue profiling in the Human Protein Atlas (HPA) project is presented. The section describing the use of protein microarrays for validation of the antibodies is relevant for this thesis. The implementation of protein microarrays in the HPA workflow was an important addition, because a deeper insight of the specificity of all the antibodies produced were now available.In a second study, bead based arrays were compared to planar protein microarrays used in the HPA project. In this study, 100 different bead identities were coupled with 100 different antigens and mixed together to generate an array. The correlation between the two types of assays was very high and the conclusion was that the methods can be used as backup to each other.A third study was a part of an international initiative to produce renewable affinity binders against proteins containing SH2 domain. Here, the HPA protein microarrays were modified to analyze different types of reagents produced at six laboratories around the world. Monoclonal antibodies, single chain fragment and fibronectin scaffolds were tested as well as mono-specific antibodies. It was shown to be possible to adapt protein microarrays used in the HPA project to validate other kinds of affinity reagents.

  • 12.
    Sundberg, Mårten
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Ramström, Margareta
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    High-abundant protein depletion strategies applied on dog cerebrospinal fluid and evaluated by high-resolution mass spectrometry2015In: Biochemistry and Biophysics Reports, ISSN 2405-5808, Vol. 3, p. 68-75Article in journal (Refereed)
    Abstract [en]

    As the number of fully sequenced animal genomes and the performance of advanced mass spectrometry-based proteomics techniques are continuously improving, there is now a great opportunity to increase the knowledge of various animal proteomes. This research area is further stimulated by a growing interest from veterinary medicine and the pharmaceutical industry. Cerebrospinal fluid (CSF) is a good source for better understanding of diseases related to the central nervous system, both in humans and other animals. In this study, four high-abundant protein depletion columns, developed for human or rat serum, were evaluated for dog CSF. For the analysis, a shotgun proteomics approach, based on nanoLC-LTQ Orbitrap MS/MS, was applied. All the selected approaches were shown to deplete dog CSF with different success. It was demonstrated that the columns significantly improved the coverage of the detected dog CSF proteome. An antibody-based column showed the best performance, in terms of efficiency, repeatability and the number of proteins detected in the sample. In total 983 proteins were detected. Of those, 801 proteins were stated as uncharacterized in the UniProt database. To the best of our knowledge, this is the so far largest number of proteins reported for dog CSF in one single study.

  • 13.
    Sundberg, Mårten
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Strage, Emma
    Sveriges lantbruksuniversitet, Institutionen för kliniska vetenskaper.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Ström Holst, Bodil
    Sveriges lantbruksuniversitet, Institutionen för kliniska vetenskaper.
    Ramström, Margareta
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Quantitative and selective analysis of feline growth related proteins using parallel reaction monitoring high resolution mass spectrometryManuscript (preprint) (Other academic)
  • 14. Uhlen, Mathias
    et al.
    Bjorling, Erik
    Agaton, Charlotta
    Szigyarto, Cristina Al-Khalili
    Amini, Bahram
    Andersen, Elisabet
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Andersson, Ann-Catrin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Angelidou, Pia
    Asplund, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Asplund, Caroline
    Berglund, Lisa
    Bergström, Kristina
    Brumer, Harry
    Cerjan, Dijana
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Ekstrom, Marica
    Elobeid, Adila
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Eriksson, Cecilia
    Fagerberg, Linn
    Falk, Ronny
    Fall, Jenny
    Forsberg, Mattias
    Björklund, Marcus Gry
    Gumbel, Kristoffer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Halimi, Asif
    Hallin, Inga
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Hamsten, Carl
    Hansson, Marianne
    Hedhammar, My
    Hercules, Görel
    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.
    Larsson, Karin
    Lindskog, Mats
    Lodewyckx, Wald
    Lund, Jan
    Lundeberg, Joakim
    Magnusson, Kristina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Malm, Erik
    Nilsson, Peter
    Odling, Jenny
    Oksvold, Per
    Olsson, Ingmarie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Oster, Emma
    Ottosson, Jenny
    Paavilainen, Linda
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Persson, Anja
    Rimini, Rebecca
    Rockberg, Johan
    Runeson, Marcus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Sivertsson, Asa
    Sköllermo, Anna
    Steen, Johanna
    Stenvall, Maria
    Sterky, Fredrik
    Strömberg, Sara
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Sundberg, Mårten
    Department of Biotechnology, AlbaNova University Center, Royal Institute of Technology (KTH), Stockholm, Sweden.
    Tegel, Hanna
    Tourle, Samuel
    Wahlund, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Waldén, Annelie
    Wan, Jinghong
    Wernéus, Henrik
    Westberg, Joakim
    Wester, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Wrethagen, Ulla
    Xu, Lan Lan
    Hober, Sophia
    Pontén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    A human protein atlas for normal and cancer tissues based on antibody proteomics2005In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 4, no 12, p. 1920-1932Article in journal (Refereed)
    Abstract [en]

    Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, ∼400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research.

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