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  • 1. Asplund, A.
    et al.
    Edqvist, P. -HD.
    Schwenk, Jochen M.
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Pontén, F.
    Antibodies for profiling the human proteome-The Human Protein Atlas as a resource for cancer research2012In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 12, no 13, 2067-2077 p.Article in journal (Refereed)
    Abstract [en]

    In this review, we present an update on the progress of the Human Protein Atlas, with an emphasis on strategies for validating immunohistochemistry-based protein expression patterns and on the possibilities to extend the map of protein expression patterns for cancer research projects. The objectives underlying the Human Protein Atlas include (i) the generation of validated antibodies toward a major isoform of all proteins encoded by the human genome, (ii) creating an information database of protein expression patterns in normal human tissues, in cells, and in cancer, and (iii) utilizing generated antibodies and protein expression data as tools to identify clinically useful biomarkers. The success of such an effort is dependent on the validity of antibodies as specific binders of intended targets in applications used to map protein expression patterns. The development of strategies to support specific target binding is crucial and remains a challenge as a large fraction of proteins encoded by the human genome is poorly characterized, including the approximately one-third of all proteins lacking evidence of existence. Conceivable methods for validation include the use of paired antibodies, i.e. two independent antibodies targeting different and nonoverlapping epitopes on the same protein as well as comparative analysis of mRNA expression patterns with corresponding proteins.

  • 2.
    Ayoglu, Burcu
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Birgersson, Elin
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Mezger, Anja
    Nilsson, Mats
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Schwenk, Jochen M.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Multiplexed protein profiling by sequential affinity capture2016In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 16, no 8, 1251-1256 p.Article in journal (Refereed)
    Abstract [en]

    Antibody microarrays enable parallelized and miniaturized analysis of clinical samples, and have proven to provide novel insights for the analysis of different proteomes. However, there are concerns that the performance of such direct labeling and single antibody assays are prone to off-target binding due to the sample context. To improve selectivity and sensitivity while maintaining the possibility to conduct multiplexed protein profiling, we developed a multiplexed and semi-automated sequential capture assay. This novel bead-based procedure encompasses a first antigen capture, labeling of captured protein targets on magnetic particles, combinatorial target elution and a read-out by a secondary capture bead array. We demonstrate in a proof-of-concept setting that target detection via two sequential affinity interactions reduced off-target contribution, while lowered background and noise levels, improved correlation to clinical values compared to single binder assays. We also compared sensitivity levels with single binder and classical sandwich assays, explored the possibility for DNA-based signal amplification, and demonstrate the applicability of the dual capture bead-based antibody microarray for biomarker analysis. Hence, the described concept enhances the possibilities for antibody array assays to be utilized for protein profiling in body fluids and beyond.

  • 3. Bendz, Maria
    et al.
    Skwark, Marcin
    Nilsson, Daniel
    Granholm, Viktor
    Cristobal, Susana
    Käll, Lukas
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Elofsson, Arne
    Membrane protein shaving with thermolysin can be used to evaluate topology predictors2013In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 13, no 9, 1467-1480 p.Article in journal (Refereed)
    Abstract [en]

    Topology analysis of membrane proteins can be obtained by enzymatic shaving in combination with MS identification of peptides. Ideally, such analysis could provide quite detailed information about the membrane spanning regions. Here, we examine the ability of some shaving enzymes to provide large-scale analysis of membrane proteome topologies. To compare different shaving enzymes, we first analyzed the detected peptides from two over-expressed proteins. Second, we analyzed the peptides from non-over-expressed Escherichia coli membrane proteins with known structure to evaluate the shaving methods. Finally, the identified peptides were used to test the accuracy of a number of topology predictors. At the end we suggest that the usage of thermolysin, an enzyme working at the natural pH of the cell for membrane shaving, is superior because: (i) we detect a similar number of peptides and proteins using thermolysin and trypsin; (ii) thermolysin shaving can be run at a natural pH and (iii) the incubation time is quite short. (iv) Fewer detected peptides from thermolysin shaving originate from the transmembrane regions. Using thermolysin shaving we can also provide a clear separation between the best and the less accurate topology predictors, indicating that using data from shaving can provide valuable information when developing new topology predictors.

  • 4.
    Berglund, Lisa
    et al.
    KTH, School of Biotechnology (BIO).
    Björling, Erik
    KTH, School of Biotechnology (BIO).
    Jonasson, Kalle
    KTH, School of Biotechnology (BIO).
    Rockberg, Johan
    KTH, School of Biotechnology (BIO).
    Fagerberg, Linn
    KTH, School of Biotechnology (BIO).
    Al-Khalili Szigyarto, Cristina
    KTH, School of Biotechnology (BIO).
    Sivertsson, Åsa
    KTH, School of Biotechnology (BIO).
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO).
    A whole-genome bioinformatics approach to selection of antigens for systematic antibody generation2008In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 8, no 14, 2832-2839 p.Article in journal (Refereed)
    Abstract [en]

    Here, we present an antigen selection strategy based on a whole-genome bioinformatics approach, which is facilitated by an interactive visualization tool displaying protein features from both public resources and in-house generated data. The web-based bioinformatics platform has been designed for selection of multiple, non-overlapping recombinant protein epitope signature tags by display of predicted information relevant for antigens, including domain- and epitope sized sequence similarities to other proteins, transmembrane regions and signal peptides. The visualization tool also displays shared and exclusive protein regions for genes with multiple splice variants. A genome-wide analysis demonstrates that antigens for approximately 80% of the human protein-coding genes can be selected with this strategy.

  • 5.
    Dezfouli, Mahya
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Vickovic, Sanja
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Iglesias, Maria Jesus
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Schwenk, Jochen M.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Magnetic bead assisted labeling of antibodies at nanogram scale2014In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 14, no 1, 14-18 p.Article in journal (Refereed)
    Abstract [en]

    There are currently several initiatives that aim to produce binding reagents for proteome-wide analysis. To enable protein detection, visualization, and target quantification, covalent coupling of reporter molecules to antibodies is essential. However, current labeling protocols recommend considerable amount of antibodies, require antibody purity and are not designed for automation. Given that small amounts of antibodies are often sufficient for downstream analysis, we developed a labeling protocol that combines purification and modification of antibodies at submicrogram quantities. With the support of magnetic microspheres, automated labeling of antibodies in parallel using biotin or fluorescent dyes was achieved.

  • 6.
    Dezfouli, Mahya
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Vickovic, Sanja
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Iglesias, Maria Jesus
    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.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Parallel barcoding of antibodies for DNA-assisted proteomics2014In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 14, no 21-22, 2432-2436 p.Article in journal (Refereed)
    Abstract [en]

    DNA-assisted proteomics technologies enable ultra-sensitive measurements in multiplex format using DNA-barcoded affinity reagents. Although numerous antibodies are available, nowadays targeting nearly the complete human proteome, the majority is not accessible at the quantity, concentration, or purity recommended for most bio-conjugation protocols. Here, we introduce a magnetic bead-assisted DNA-barcoding approach, applicable for several antibodies in parallel, as well as reducing required reagents quantities up to a thousand-fold. The success of DNA-barcoding and retained functionality of antibodies were demonstrated in sandwich immunoassays and standard quantitative Immuno-PCR assays. Specific DNA-barcoding of antibodies for multiplex applications was presented on suspension bead arrays with read-out on a massively parallel sequencing platform in a procedure denoted Immuno-Sequencing. Conclusively, human plasma samples were analyzed to indicate the functionality of barcoded antibodies in intended proteomics applications.

  • 7.
    Fagerberg, Linn
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Jonasson, Kalle
    KTH, School of Biotechnology (BIO), Proteomics.
    von Heijne, Gunnar
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Berglund, Lisa
    KTH, School of Biotechnology (BIO), Proteomics.
    Prediction of the human membrane proteome2010In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 10, no 6, 1141-1149 p.Article in journal (Refereed)
    Abstract [en]

    Membrane proteins are key molecules in the cell, and are important targets for pharmaceutical drugs. Few three-dimensional structures of membrane proteins have been obtained, which makes computational prediction of membrane proteins crucial for studies of these key molecules. Here, seven membrane protein topology prediction methods based on different underlying algorithms, such as hidden Markov models, neural networks and support vector machines, have been used for analysis of the protein sequences from the 21 416 annotated genes in the human genome. The number of genes coding for a protein with predicted cc-helical transmembrane region(s) ranged from 5508 to 7651, depending on the method used. Based on a majority decision method, we estimate 5539 human genes to code for membrane proteins, corresponding to approximately 26% of the human protein-coding genes. The largest fraction of these proteins has only one predicted transmembrane region, but there are also many proteins with seven predicted transmembrane regions, including the G-protein coupled receptors. A visualization tool displaying the topologies suggested by the eight prediction methods, for all predicted membrane proteins, is available on the public Human Protein Atlas portal (www.proteinatlas.org).

  • 8. Granholm, Viktor
    et al.
    Käll, Lukas
    Quality assessments of peptide-spectrum matches in shotgun proteomics2011In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 11, no 6, 1086-1093 p.Article in journal (Refereed)
    Abstract [en]

    The peptide identification process in shotgun proteomics is most frequently solved with search engines. Such search engines assign scores that reflect similarity between the measured fragmentation spectrum and the theoretical spectra of the peptides of a given database. However, the scores from most search engines do not have a direct statistical interpretation. To understand and make use of the significance of peptide identifications, one must thus be familiar with some statistical concepts. Here, we discuss different statistical scores used to show the confidence of an identification and a set of methods to estimate these scores. We also describe the variance of statistical scores and imperfections of scoring functions of peptide-spectrum matches.

  • 9.
    Häggmark, Anna
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Byström, Sanna
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ayoglu, Burcu
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Qundos, Ulrika
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Khademi, M.
    Olsson, T.
    Schwenk, Jochen M.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nilsson, Peter
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Antibody-based profiling of cerebrospinal fluid within multiple sclerosis2013In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 13, no 15, 2256-2267 p.Article in journal (Refereed)
    Abstract [en]

    Antibody suspension bead arrays have proven to enable multiplexed and high-throughput protein profiling in unfractionated plasma and serum samples through a direct labeling approach. We here describe the development and application of an assay for protein profiling of cerebrospinal fluid (CSF). While setting up the assay, systematic intensity differences between sample groups were observed that reflected inherent sample specific total protein amounts. Supplementing the labeling reaction with BSA and IgG diminished these differences without impairing the apparent sensitivity of the assay. We also assessed the effects of heat treatment on the analysis of CSF proteins and applied the assay to profile 43 selected proteins by 101 antibodies in 339 CSF samples from a multiple sclerosis (MS) cohort. Two proteins, GAP43 and SERPINA3 were found to have a discriminating potential with altered intensity levels between sample groups. GAP43 was detected at significantly lower levels in secondary progressive MS compared to early stages of MS and the control group of other neurological diseases. SERPINA3 instead was detected at higher levels in all MS patients compared to controls. The developed assay procedure now offers new possibilities for broad-scale protein profiling of CSF within neurological disorders.

  • 10.
    Jonasson, Kalle
    et al.
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Berglund, Lisa
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    The 6th HUPO Antibody Initiative (HAI) workshop: Sharing data about affinity reagents and other recent developments2010In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 10, no 11, 2066-2068 p.Article in journal (Refereed)
    Abstract [en]

    The Human Antibody Initiative (HAI) aims to promote and facilitate the use of antibodies for proteomics research. The 6th workshop for the HUPO Antibody Initiative (HAI) held in September 2009 was co-chaired by Michael Snyder and Mathias Uhlen and discussed several aspects of antibody production, their validation, and attempts to standardise this process, in particular, when subsequently described in the literature. An update on the progress of the Human Protein Atlas was also presented to the attendees.

  • 11. Kampf, Caroline
    et al.
    Mardinoglu, Adil
    Fagerberg, Linn
    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.
    Danielsson, Angelika
    Nielsen, Jens
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Chalmers University of Technology, Sweden.
    Pontén, Fredrik
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Defining the human gallbladder proteome by transcriptomics and affinity proteomics2014In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 14, no 21-22, 2498-2507 p.Article in journal (Refereed)
    Abstract [en]

    Global protein analysis of human gallbladder tissue is vital for identification of molecular regulators and effectors of its physiological activity. Here, we employed a genome-wide deep RNA sequencing analysis in 28 human tissues to identify the genes overrepresented in the gallbladder and complemented it with antibody-based immunohistochemistry in 48 human tissues. We characterized human gallbladder proteins and identified 140 gallbladder-specific proteins with an elevated expression in the gallbladder as compared to the other analyzed tissues. Five genes were categorized as enriched, with at least fivefold higher levels in gallbladder, 60 genes were categorized as group enriched with elevated transcript levels in gallbladder shared with at least one other tissue and 75 genes were categorized as enhanced with higher expression than the average expression in other tissues. We explored the localization of the genes within the gallbladder through cell-type specific antibody-based protein profiling and the subcellular localization of the genes through immunofluorescent-based profiling. Finally, we revealed the biological processes and metabolic functions carried out by these genes through the use of GO, KEGG Pathway, and HMR2.0 that is compilation of the human metabolic reactions. We demonstrated the results of the combined analysis of the transcriptomics and affinity proteomics.

  • 12. Lal, S.
    et al.
    Nguyen, L.
    Tezone, R.
    Pontén, Fredrik
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Odeberg, J.
    Li, A.
    dos Remedios, C.
    Tissue microarray profiling in human heart failure2016In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861Article in journal (Refereed)
    Abstract [en]

    Tissue MicroArrays (TMAs) are a versatile tool for high-throughput protein screening, allowing qualitative analysis of a large number of samples on a single slide. We have developed a customizable TMA system that uniquely utilizes cryopreserved human cardiac samples from both heart failure and donor patients to produce formalin-fixed paraffin-embedded sections. Confirmatory upstream or downstream molecular studies can then be performed on the same (biobanked) cryopreserved tissue. In a pilot study, we applied our TMAs to screen for the expression of four-and-a-half LIM-domain 2 (FHL2), a member of the four-and-a-half LIM family. This protein has been implicated in the pathogenesis of heart failure in a variety of animal models. While FHL2 is abundant in the heart, not much is known about its expression in human heart failure. For this purpose, we generated an affinity-purified rabbit polyclonal anti-human FHL2 antibody. Our TMAs allowed high-throughput profiling of FHL2 protein using qualitative and semiquantitative immunohistochemistry that proved complementary to Western blot analysis. We demonstrated a significant relative reduction in FHL2 protein expression across different forms of human heart failure.

  • 13.
    Lundberg, Emma
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Creation of an antibody-based subcellular protein atlas2010In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 10, no 22, 3984-3996 p.Article, review/survey (Refereed)
    Abstract [en]

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

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

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

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

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

  • 16.
    Neiman, Maja
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Fredolini, Claudia
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Johansson, H.
    Lehtiö, Janne
    Karolinska Institutet, Solna, Sweden .
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Protein Technology.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Schwenk, Jochen M.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Selectivity analysis of single binder assays used in plasma protein profiling2013In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 13, no 23-24, 3406-3410 p.Article in journal (Refereed)
    Abstract [en]

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

  • 17.
    Nilsson, Peter
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Larsson, Karin
    KTH, School of Biotechnology (BIO).
    Persson, Anja
    KTH, School of Biotechnology (BIO), Proteomics.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Wernérus, Henrik
    KTH, School of Biotechnology (BIO).
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Björling, Erik
    KTH, School of Biotechnology (BIO).
    Ottoson, Jenny
    KTH, School of Biotechnology (BIO), Proteomics.
    Ödling, Jenny
    KTH, School of Biotechnology (BIO).
    Sundberg, Mårten
    KTH, School of Biotechnology (BIO), Proteomics.
    Al-Khalili Szigyarto, Cristina
    KTH, School of Biotechnology (BIO), Proteomics.
    Paavilainen, Linda
    Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
    Andersson, Ann-Catrin
    Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
    Kampf, Caroline
    Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
    Wester, Kenneth
    Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
    Pontén, Fredrik
    Department of Genetics and Pathology, The Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
    Towards a human proteome atlas: High-throughput generation of mono-specific antibodies for tissue profiling2005In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 5, 4327-4337 p.Article 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.

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

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

  • 19.
    Pang, Zhili
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience. China Agricultural University, China.
    Chen, Lei
    Miao, Jianqiang
    Wang, Zhiwen
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience. University of Adelaide, Australia.
    Liu, Xili
    Proteomic profile of the plant-pathogenic oomycete Phytophthora capsici in response to the fungicide pyrimorph2015In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 15, no 17, 2972-2982 p.Article in journal (Refereed)
    Abstract [en]

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

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

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

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

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

  • 22.
    Schwenk, Jochen M.
    et al.
    KTH, School of Biotechnology (BIO), Proteomics.
    Igel, Ulrika
    KTH, School of Biotechnology (BIO), Proteomics.
    Kato, Bernet S.
    Nicholson, George
    Karpe, Fredrik
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics.
    Comparative protein profiling of serum and plasma using an antibody suspension bead array approach2010In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 10, no 3, 532-540 p.Article in journal (Refereed)
    Abstract [en]

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

  • 23. Sialana, Fernando J.
    et al.
    Gulyassy, Peter
    Majek, Peter
    Sjöstedt, Evelina
    KTH, Centres, Science for Life Laboratory, SciLifeLab. Uppsala University, Sweden.
    Kis, Viktor
    Muller, Andre C.
    Rudashevskaya, Elena L.
    Mulder, Jan
    Bennett, Keiryn L.
    Lubec, Gert
    Mass spectrometric analysis of synaptosomal membrane preparations for the determination of brain receptors, transporters and channels2016In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 16, no 22, 2911-2920 p.Article in journal (Refereed)
    Abstract [en]

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

  • 24.
    Strömberg, Sara
    et al.
    Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University.
    Gry Björklund, Marcus
    KTH, School of Biotechnology (BIO), Proteomics.
    Asplund, Caroline
    KTH, School of Biotechnology (BIO), Proteomics.
    Sköllermo, Anna
    KTH, School of Biotechnology (BIO), Proteomics.
    Persson, Anja
    KTH, School of Biotechnology (BIO), Proteomics.
    Wester, Kenneth
    Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University.
    Kampf, Caroline
    Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics.
    Andersson, Ann-Catrin
    Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Kononen, Juha
    Beecher Instruments, Sun Prairie, WI, United States.
    Pontén, Fredrik
    Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University.
    Asplund, Anna
    Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University.
    A high-throughput strategy for protein profiling in cell microarrays using automated image analysis2007In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 7, no 13, 2142-2150 p.Article in journal (Refereed)
    Abstract [en]

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

  • 25.
    The, Matthew
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Tasnim, Ayesha
    KTH, School of Biotechnology (BIO), Gene Technology.
    Käll, Lukas
    KTH, School of Biotechnology (BIO), Gene Technology.
    How to talk about protein-level false discovery rates in shotgun proteomics2016In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 16, no 18, 2461-2469 p.Article in journal (Refereed)
    Abstract [en]

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

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

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

1 - 26 of 26
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