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  • 1. Baan, Bart
    et al.
    Pardali, Evangelia
    ten Dijke, Peter
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwiginstitutet för cancerforskning.
    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 vivo2010Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 9, nr 9, s. 1982-1990Artikel i tidskrift (Refereegranskat)
    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.

  • 2. Barbe, Laurent
    et al.
    Lundberg, Emma
    Oksvold, Per
    Stenius, Anna
    Lewin, Erland
    Björling, Erik
    Asplund, Anna
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Pontén, Fredrik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Brismar, Hjalmar
    Uhlén, Mathias
    Andersson-Svahn, Helene
    Toward a confocal subcellular atlas of the human proteome2008Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 7, nr 3, s. 499-508Artikel i tidskrift (Refereegranskat)
    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.

  • 3.
    Berggrund, Malin
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Medicinsk genetik och genomik. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Enroth, Stefan
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, 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 universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för kvinnors och barns hälsa, Forskargrupper (Inst. för kvinnor och barns hälsa), Reproduktiv hälsa.
    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 universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för kvinnors och barns hälsa, Forskargrupper (Inst. för kvinnor och barns hälsa), Reproduktionsbiologi.
    Gyllensten, Ulf
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Medicinsk genetik och genomik.
    Identification of Candidate Plasma Protein Biomarkers for Cervical Cancer Using the Multiplex Proximity Extension Assay2019Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 18, nr 4, s. 735-743Artikel i tidskrift (Refereegranskat)
    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.

  • 4. Berglund, Lisa
    et al.
    Björling, Erik
    Oksvold, Per
    Fagerberg, Linn
    Asplund, Anna
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Szigyarto, Cristina Al-Khalili
    Persson, Anja
    Ottosson, Jenny
    Wernérus, Henrik
    Nilsson, Peter
    Lundberg, Emma
    Sivertsson, Åsa
    Navani, Sanjay
    Wester, Kenneth
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Kampf, Caroline
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Hober, Sophia
    Pontén, Fredrik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Uhlén, Mathias
    A genecentric Human Protein Atlas for expression profiles based on antibodies2008Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 7, nr 10, s. 2019-2027Artikel, forskningsöversikt (Refereegranskat)
    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.

  • 5.
    Björkesten, Johan
    et al.
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi.
    Enroth, Stefan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Shen, Qiujin
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi.
    Wik, Lotta
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi. Uppsala universitet, 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 universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för folkhälso- och vårdvetenskap, Geriatrik.
    Ingelsson, Martin
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för folkhälso- och vårdvetenskap, Geriatrik.
    Larsson, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper.
    Kamali-Moghaddam, Masood
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Landegren, Ulf
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Stability of Proteins in Dried Blood Spot Biobanks.2017Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 16, nr 7, s. 1286-1296Artikel i tidskrift (Refereegranskat)
    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.

  • 6. Björling, Erik
    et al.
    Lindskog, Cecilia
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Oksvold, Per
    Linné, Jerker
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Kampf, Caroline
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Hober, Sophia
    Uhlén, Mathias
    Pontén, Fredrik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    A web-based tool for in silico biomarker discovery based on tissue-specific protein profiles in normal and cancer tissues2008Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 7, nr 5, s. 825-844Artikel i tidskrift (Refereegranskat)
    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.

  • 7. Chornoguz, Olesya
    et al.
    Grmai, Lydia
    Sinha, Pratima
    Artemenko, Konstantin A.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för fysikalisk och analytisk kemi, Analytisk kemi.
    Zubarev, Roman A.
    Ostrand-Rosenberg, Suzanne
    Proteomic Pathway Analysis Reveals Inflammation Increases Myeloid-Derived Suppressor Cell Resistance to Apoptosis2011Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 10, nr 3Artikel i tidskrift (Refereegranskat)
    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.

  • 8. 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 universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Sun, Wei
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Dell, Anne
    Tumor Biomarker Glycoproteins in the Seminal Plasma of Healthy Human Males Are Endogenous Ligands for DC-SIGN2012Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 11, nr 1Artikel i tidskrift (Refereegranskat)
    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.

  • 9. 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.2006Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 5, nr 10, s. 1975-83Artikel i tidskrift (Refereegranskat)
    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.

  • 10.
    Darmanis, Spyros
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Nong, Rachel Yuan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Hammond, Maria
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Gu, Jijuan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Alderborn, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Vänelid, Johan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Siegbahn, Agneta
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper.
    Gustafsdottir, Sigrun
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Ericsson, Olle
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Landegren, Ulf
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Kamali-Moghaddam, Masood
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Sensitive plasma protein analysis by microparticle-based proximity ligation assays2010Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 9, nr 2, s. 327-335Artikel i tidskrift (Refereegranskat)
    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.

  • 11.
    Dubois, Louise
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Biokemisk struktur och funktion.
    Ronquist, K Göran
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Biokemisk struktur och funktion.
    Ek, Bo
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Analytisk kemi.
    Ronquist, Gunnar
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Biokemisk struktur och funktion.
    Larsson, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Biokemisk struktur och funktion.
    Proteomic profiling of detergent resistant membranes (lipid rafts) of prostasomes2015Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 14, nr 11, s. 3015-3022Artikel i tidskrift (Refereegranskat)
    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.

  • 12. Fagerberg, Linn
    et al.
    Hallström, Björn M
    Oksvold, Per
    Kampf, Caroline
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi.
    Djureinovic, Dijana
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi.
    Odeberg, Jacob
    Habuka, Masato
    Tahmasebpoor, Simin
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi.
    Danielsson, Angelika
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi.
    Edlund, Karolina
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi.
    Asplund, Anna
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi.
    Sjöstedt, Evelina
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi.
    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 universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi.
    Danielsson, Frida
    Mardinoglu, Adil
    Sivertsson, Asa
    von Feilitzen, Kalle
    Forsberg, Mattias
    Zwahlen, Martin
    Olsson, IngMarie
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi.
    Navani, Sanjay
    Huss, Mikael
    Nielsen, Jens
    Pontén, Fredrik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi.
    Uhlén, Mathias
    Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics2014Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 13, nr 2, s. 397-406Artikel i tidskrift (Refereegranskat)
    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.

  • 13.
    Fälth, Maria
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap, MMS, medicinsk masspektrometri.
    Sköld, Karl
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap, MMS, medicinsk masspektrometri.
    Norrman, Mathias
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap, MMS, medicinsk masspektrometri.
    Svensson, Marcus
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap, MMS, medicinsk masspektrometri.
    Fenyö, David
    Andrén, Per E
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap, MMS, medicinsk masspektrometri.
    SwePep – A database designed for endogenous peptides and mass spectrometry2006Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 5, nr 6, s. 998-1005Artikel i tidskrift (Refereegranskat)
    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.

  • 14.
    Fälth, Maria
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap, MMS, medicinsk masspektrometri.
    Sköld, Karl
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap, MMS, medicinsk masspektrometri.
    Norrman, Mathias
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap, MMS, medicinsk masspektrometri.
    Svensson, Marcus
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap, MMS, medicinsk masspektrometri.
    Fenyö, David
    Andrén, Per E
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap, MMS, medicinsk masspektrometri.
    SwePep, a database designed for endogenous peptides and mass spectrometry2006Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 5, nr 6, s. 998-1005Artikel i tidskrift (Refereegranskat)
    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.

  • 15.
    Fälth, Maria
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap, MMS, medicinsk masspektrometri.
    Sköld, Karl
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap, MMS, medicinsk masspektrometri.
    Svensson, Marcus
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap, MMS, medicinsk masspektrometri.
    Nilsson, Anna
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap, MMS, medicinsk masspektrometri.
    Fenyö, David
    Andrén, Per E.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap, MMS, medicinsk masspektrometri.
    Neuropeptidomics strategies for specific and sensitive identification of endogenous peptides2007Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 6, nr 7, s. 1188-1197Artikel i tidskrift (Refereegranskat)
    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.

  • 16. Geiger, Tamar
    et al.
    Velic, Ana
    Macek, Boris
    Lundberg, Emma
    Kampf, Caroline
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi.
    Nagaraj, Nagarjuna
    Uhlen, Mathias
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi.
    Cox, Juergen
    Mann, Matthias
    Initial quantitative proteomic map of 28 mouse tissues using the SILAC mouse2013Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 12, nr 6, s. 1709-1722Artikel i tidskrift (Refereegranskat)
    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.

  • 17. Gloriam, David E.
    et al.
    Orchard, Sandra
    Bertinetti, Daniela
    Björling, Erik
    Bongcam-Rudloff, Erik
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Centrum för bioinformatik.
    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 universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    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 Reagents2010Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 9, nr 1, s. 1-10Artikel i tidskrift (Refereegranskat)
    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.

  • 18.
    Hanrieder, Jörg
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för fysikalisk och analytisk kemi, Analytisk kemi. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap.
    Ljungdahl, Anna
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap.
    Fälth, Maria
    Eriksson Mammo, Sofie
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap.
    Bergquist, Jonas
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för fysikalisk och analytisk kemi, Analytisk kemi.
    Andersson, Malin
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap.
    L-DOPA-induced dyskinesia is associated with regional increase of striatal dynorphin peptides as elucidated by imaging mass spectrometry2011Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 10, nr 10, s. M111.009308-Artikel i tidskrift (Refereegranskat)
    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.

  • 19.
    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 proteins2002Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 1, nr 12, s. 956-966Artikel i tidskrift (Refereegranskat)
    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.

  • 20. 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 levels2005Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 4, nr 12, s. 1942-1947Artikel i tidskrift (Refereegranskat)
    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.

  • 21.
    Jarvius, Malin
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Paulsson, Janna
    Weibrecht, Irene
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Leuchowius, Karl-Johan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Andersson, Ann-Catrin
    Wählby, Carolina
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Matematisk-datavetenskapliga sektionen, Institutionen för informationsteknologi, Datoriserad bildanalys. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Matematisk-datavetenskapliga sektionen, Centrum för bildanalys.
    Gullberg, Mats
    Botling, Johan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Sjöblom, Tobias
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Markova, Boyka
    Östman, Arne
    Landegren, Ulf
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Söderberg, Ola
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    In situ detection of phosphorylated platelet-derived growth factor receptor beta using a generalized proximity ligation method2007Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 6, nr 9, s. 1500-1509Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Improved methods are needed for in situ characterization of post-translational modifications in cell lines and tissues. For example, it is desirable to monitor the phosphorylation status of individual receptor tyrosine kinases in samples from human tumors treated with inhibitors to evaluate therapeutic responses. Unfortunately the leading methods for observing the dynamics of tissue post-translational modifications in situ, immunohistochemistry and immunofluorescence, exhibit limited sensitivity and selectivity. Proximity ligation assay is a novel method that offers improved selectivity through the requirement of dual recognition and increased sensitivity by including DNA amplification as a component of detection of the target molecule. Here we therefore established a generalized in situ proximity ligation assay to investigate phosphorylation of platelet-derived growth factor receptor β (PDGFRβ) in cells stimulated with platelet-derived growth factor BB. Antibodies specific for immunoglobulins from different species, modified by attachment of DNA strands, were used as secondary proximity probes together with a pair of primary antibodies from the corresponding species. Dual recognition of receptors and phosphorylated sites by the primary antibodies in combination with the secondary proximity probes was used to generate circular DNA strands; this was followed by signal amplification by replicating the DNA circles via rolling circle amplification. We detected tyrosine phosphorylated PDGFRβ in human embryonic kidney cells stably overexpressing human influenza hemagglutinin-tagged human PDGFRβ in porcine aortic endothelial cells transfected with the β-receptor, but not in cells transfected with the α-receptor, and also in immortalized human foreskin fibroblasts, BJ hTert, endogenously expressing the PDGFRβ. We furthermore visualized tyrosine phosphorylated PDGFRβ in tissue sections from fresh frozen human scar tissue undergoing wound healing. The method should be of great value to study signal transduction, screen for effects of pharmacological agents, and enhance the diagnostic potential in histopathology.

  • 22.
    Karlsson, Oskar
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap.
    Bergquist, Jonas
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Analytisk kemi.
    Andersson, Malin
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap.
    Quality measures of imaging mass spectrometry aids in revealing long-term striatal protein changes induced by neonatal exposure to the cyanobacterial toxin β-N-methylamino-L-alanine (BMAA)2014Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 13, s. 93-104Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Many pathological processes are not directly correlated to dramatic alterations in protein levels. The changes in local concentration of important proteins in a subset of cells or at specific loci is likely to play a significant role in the disease etiologies, but the precise location might be unknown or too small to be adequately sampled for the purpose of traditional proteomic techniques. In this respect, matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) is a unique analytical method that combines analysis of multiple molecular species and their distribution in one single platform. As reproducibility is essential for successful biomarker discovery it is important to systemically assess data quality in biologically relevant MALDI IMS experiments. In the present study, we applied four simple tools to study the reproducibility for individual sections, within group variation and between group variations of data acquired from brain sections of 21 animals divided into three treatment groups. We also characterized protein changes in distinct regions of the striatum from six month-old rats treated neonatally (PND; postnatal days 9-10) with the cyanobacterial toxin β-N-methylamino-L-alanine (BMAA) that has been implicated in neurodegenerative diseases. The results showed that optimized experimental settings can render high quality MALDI IMS data with relatively low variation (14-15 %CV) that allows characterization of subtle changes in protein expression in various subregions of the brain. This was further exemplified by the dose-dependent reduction of MBP (myelin basic protein) in the caudate putamen and the nucleus accumbens of adult rats neonatally treated with BMAA (150 and 460 mg/kg). The MBP reduction was confirmed with immunohistochemistryand indicates that developmental exposure to BMAA may induce structural effects on axonal growth and/or directly on proliferation of oligodendrocytes and myelination, which might be important for the previously shown BMAA-induced long-term cognitive impairments.

  • 23.
    Kultima, Kim
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Klinisk farmakologi.
    Nilsson, Anna
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap.
    Scholz, Birger
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap.
    Rossbach, Uwe L.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap.
    Fälth, Maria
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap.
    Andrén, Per E.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap.
    Development and evaluation of normalization methods for label-free relative quantification of endogenous peptides2009Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 8, nr 10, s. 2285-2295Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The performances of 10 different normalization methods on data of endogenous brain peptides produced with label-free nano-LC-MS were evaluated. Data sets originating from three different species (mouse, rat, and Japanese quail), each consisting of 35-45 individual LC-MS analyses, were used in the study. Each sample set contained both technical and biological replicates, and the LC-MS analyses were performed in a randomized block fashion. Peptides in all three data sets were found to display LC-MS analysis order-dependent bias. Global normalization methods will only to some extent correct this type of bias. Only the novel normalization procedure RegrRun (linear regression followed by analysis order normalization) corrected for this type of bias. The RegrRun procedure performed the best of the normalization methods tested and decreased the median S.D. by 43% on average compared with raw data. This method also produced the smallest fraction of peptides with interblock differences while producing the largest fraction of differentially expressed peaks between treatment groups in all three data sets. Linear regression normalization (Regr) performed second best and decreased median S.D. by 38% on average compared with raw data. All other examined methods reduced median S.D. by 20-30% on average compared with raw data.

  • 24. Larssen, Pia
    et al.
    Wik, Lotta
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi.
    Czarnewski, Paulo
    Eldh, Maria
    Löf, Liza
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi.
    Ronquist, K Göran
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper.
    Dubois, Louise
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper.
    Freyhult, Eva
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper.
    Gallant, Caroline J
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi.
    Oelrich, Johan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi.
    Larsson, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper.
    Ronquist, Gunnar
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper.
    Villablanca, Eduardo J
    Landegren, Ulf
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi.
    Gabrielsson, Susanne
    Kamali-Moghaddam, Masood
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi.
    Tracing Cellular Origin of Human Exosomes Using Multiplex Proximity Extension Assays.2017Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 16, nr 8, artikel-id 1547Artikel i tidskrift (Refereegranskat)
  • 25.
    Lee, Chien-Yun
    et al.
    Natl Chung Hsing Univ, Taipei, Taiwan;Acad Sinica, Mol & Biol Agr Sci Program, Taiwan Int Grad Program, Taipei, Taiwan;Natl Chung Hsing Univ, Grad Inst Biotechnol, Taichung, Taiwan;Tech Univ Munich, Chair Prote & Bioanalyt, Emil Erlenmeyer Forum 5, D-85354 Freising Weihenstephan, Germany.
    Wang, Dongxue
    Tech Univ Munich, Chair Prote & Bioanalyt, Emil Erlenmeyer Forum 5, D-85354 Freising Weihenstephan, Germany.
    Wilhelm, Mathias
    Tech Univ Munich, Chair Prote & Bioanalyt, Emil Erlenmeyer Forum 5, D-85354 Freising Weihenstephan, Germany.
    Zolg, Daniel P.
    Tech Univ Munich, Chair Prote & Bioanalyt, Emil Erlenmeyer Forum 5, D-85354 Freising Weihenstephan, Germany.
    Schmidt, Tobias
    Tech Univ Munich, Chair Prote & Bioanalyt, Emil Erlenmeyer Forum 5, D-85354 Freising Weihenstephan, Germany.
    Schnatbaum, Karsten
    JPT Peptide Technol GmbH, Berlin, Germany.
    Reimer, Ulf
    JPT Peptide Technol GmbH, Berlin, Germany.
    Pontén, Fredrik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Klinisk och experimentell patologi.
    Uhlen, Mathias
    KTH Royal Inst Technol, Sci Life Lab, Stockholm, Sweden.
    Hahne, Hannes
    OmicScouts GmbH, Freising Weihenstephan, Germany.
    Kuster, Bernhard
    Natl Chung Hsing Univ, Taipei, Taiwan;Bavarian Ctr Biomol Mass Spectrometry, Freising Weihenstephan, Germany;Tech Univ Munich, Chair Prote & Bioanalyt, Emil Erlenmeyer Forum 5, D-85354 Freising Weihenstephan, Germany.
    Mining the Human Tissue Proteome for Protein Citrullination2018Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 17, nr 7, s. 1378-1391Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 26.
    Leuchowius, Karl-Johan
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Clausson, Carl-Magnus
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Grannas, Karin
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Erbilgin, Yücel
    Botling, Johan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Zieba, Agata
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Landegren, Ulf
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Söderberg, Ola
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Parallel Visualization of Multiple Protein Complexes in Individual Cells in Tumor Tissue2013Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 12, nr 6, s. 1563-1571Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Cellular functions are regulated and executed by complex protein interaction networks. Accordingly, it is essential to understand the interplay between proteins in determining the activity status of signaling cascades. New methods are therefore required to provide information on different protein interaction events at the single cell level in heterogeneous cell populations such as in tissue sections. Here, we describe a multiplex proximity ligation assay for simultaneous visualization of multiple protein complexes in situ. The assay is an enhancement of the original proximity ligation assay, and it is based on using proximity probes labeled with unique tag sequences that can be used to read out which probes, from a pool of probes, have bound a certain protein complex. Using this approach, it is possible to gain information on the constituents of different protein complexes, the subcellular location of the complexes, and how the balance between different complex constituents can change between normal and malignant cells, for example. As a proof of concept, we used the assay to simultaneously visualize multiple protein complexes involving EGFR, HER2, and HER3 homo- and heterodimers on a single-cell level in breast cancer tissue sections. The ability to study several protein complex formations concurrently at single cell resolution could be of great potential for a systems understanding, paving the way for improved disease diagnostics and possibilities for drug development.

  • 27.
    Leuchowius, Karl-Johan
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Jarvius, Malin
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Wickström, Malin
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Klinisk farmakologi.
    Rickardson, Linda
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Klinisk farmakologi.
    Landegren, Ulf
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Larsson, Rolf
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Klinisk farmakologi.
    Söderberg, Ola
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Fryknäs, Mårten
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Klinisk farmakologi.
    Jarvius, Jonas
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    High content screening for inhibitors of protein interactions and post-translational modifications in primary cells by proximity ligation2010Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 9, nr 1, s. 178-183Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The cost of developing new drugs is a major obstacle for pharmaceutical companies and academia with many drugs identified in the drug discovery process failing approval for clinical use due to lack of intended effect or because of severe side effects. Since the early 1990 s, high throughput screening of drug compounds has increased enormously in capacity but has not resulted in a higher success rate of the identified drugs. Thus, there is a need for methods that can identify biologically relevant compounds and more accurately predict in vivo effects early in the drug discovery process. To address this, we developed a proximity ligation-based assay for high content screening of drug effects on signaling pathways. As a proof of concept, we used the assay to screen through a library of previously identified kinase inhibitors, including six clinically used tyrosine kinase inhibitors, to identify compounds that inhibited the platelet-derived growth factor (PDGF) receptor beta signaling pathway in stimulated primary human fibroblasts. Thirteen of the 80 compounds were identified as hits, and the dose responses of these compounds were measured. The assay exhibited a very high Z' factor (0.71) and signal to noise ratio (11.7), demonstrating excellent ability to identify compounds interfering with the specific signaling event. A comparison with regular immunofluorescence detection of phosphorylated PDGF receptor demonstrated a far superior ability by the in situ proximity ligation assay to reveal inhibition of receptor phosphorylation. In addition, inhibitor-induced perturbation of protein-protein interactions of the PDGF signaling pathway could be quantified, further demonstrating the usefulness of the assay in drug discovery.

  • 28.
    Liu, Yanling
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Gu, Jijuan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Hagner-McWhirter, Asa
    Sathiyanarayanan, Poojahrau
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Gullberg, Mats
    Söderberg, Ola
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Johansson, Johan
    Hammond, Maria
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Ivansson, Daniel
    Landegren, Ulf
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Western Blotting via Proximity Ligation for High Performance Protein Analysis2011Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 10, nr 11, s. O111.011031-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Western blotting is a powerful and widely used method, but limitations in detection sensitivity and specificity, and dependence upon high quality antibodies to detect targeted proteins, are hurdles to overcome. The in situ proximity ligation assay, based on dual antibody recognition and powerful localized signal amplification, offers increased detection sensitivity and specificity, along with an ability to identify complex targets such as phosphorylated or interacting proteins. Here we have applied the in situ proximity ligation assay mechanism in Western blotting. This combination allowed the use of isothermal rolling circle amplification of DNA molecules formed in target-specific ligation reaction, for 16-fold or greater increase in detection sensitivity. The increased specificity because of dual antibody recognition ensured highly selective assays, detecting the specific band when combinations of two cross-reactive antitubulin antibodies were used (i.e. both producing distinct nonspecific bands in traditional Western blotting). We also demonstrated detection of phosphorylated platelet-derived growth factor receptor beta by proximity ligation with one antibody directed against the receptor and another directed against the phosphorylated tyrosine residue. This avoided the need for stripping and re-probing the membrane or aligning two separate traditional blots. We demonstrate that the high-performance in situ proximity ligation-based Western blotting described herein is compatible with detection via enhanced chemiluminescence and fluorescence detection systems, and can thus be readily employed in any laboratory.

  • 29. Lundberg, Martin
    et al.
    Thorsen, Stine Buch
    Assarsson, Erika
    Villablanca, Andrea
    Tran, Bonnie
    Gee, Nick
    Knowles, Mick
    Nielsen, Birgitte Sander
    Gonzalez Couto, Eduardo
    Martin, Roberto
    Nilsson, Olle
    Fermer, Christian
    Schlingemann, Joerg
    Christensen, Ib Jarle
    Nielsen, Hans-Jorgen
    Ekström, Björn
    Andersson, Claes
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper.
    Gustafsson, Mats
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper.
    Brunner, Nils
    Stenvang, Jan
    Fredriksson, Simon
    Multiplexed Homogeneous Proximity Ligation Assays for High-throughput Protein Biomarker Research in Serological Material2011Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 10, nr 4, s. M110.004978-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A high throughput protein biomarker discovery tool has been developed based on multiplexed proximity ligation assays in a homogeneous format in the sense of no washing steps. The platform consists of four 24-plex panels profiling 74 putative biomarkers with sub-pM sensitivity each consuming only 1 mu l of human plasma sample. The system uses either matched monoclonal antibody pairs or the more readily available single batches of affinity purified polyclonal antibodies to generate the target specific reagents by covalently linking with unique nucleic acid sequences. These paired sequences are united by DNA ligation upon simultaneous target binding forming a PCR amplicon. Multiplex proximity ligation assays thereby converts multiple target analytes into real-time PCR amplicons that are individually quantified using microfluidic high capacity qPCR in nano liter volumes. The assay shows excellent specificity, even in multiplex, by its dual recognition feature, its proximity requirement, and most importantly by using unique sequence specific reporter fragments on both antibody-based probes. To illustrate the potential of this protein detection technology, a pilot biomarker research project was performed using biobanked plasma samples for the detection of colorectal cancer using a multivariate signature.

  • 30.
    Månberg, Anna
    et al.
    KTH Royal Inst Technol, Dept Prot Sci, SciLifeLab, Affin Prote, Stockholm, Sweden.
    Bradley, Frideborg
    Karolinska Univ Hosp, Ctr Mol Med, Karolinska Inst, Dept Med Solna,Unit Infect Dis, Stockholm, Sweden.
    Qundos, Ulrika
    KTH Royal Inst Technol, Dept Prot Sci, SciLifeLab, Affin Prote, Stockholm, Sweden.
    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 universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Klinisk och experimentell patologi.
    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 Royal Inst Technol, Dept Prot Sci, SciLifeLab, Affin Prote, Stockholm, Sweden.
    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 Infection2019Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 18, nr 3, s. 461-476Artikel i tidskrift (Refereegranskat)
    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.

  • 31.
    Namuduri, Arvind Venkat
    et al.
    Karolinska Inst.
    Heras, Gabriel
    Karolinska Inst.
    Mi, Jia
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Analytisk kemi. Uppsala universitet, Science for Life Laboratory, SciLifeLab. Binzhou Med Univ, Peoples R China..
    Cacciani, Nicola
    Karolinska Inst.
    Hörnaeus, Katarina
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Analytisk kemi.
    Konzer, Anne
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Analytisk kemi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Bergström Lind, Sara
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Analytisk kemi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Larsson, Lars
    Karolinska Inst.
    Gastaldello, Stefano
    Karolinska Inst.
    A Proteomic Approach to Identify Alterations in the Small Ubiquitin-like Modifier (SUMO) Network during Controlled Mechanical Ventilation in Rat Diaphragm Muscle2017Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 16, nr 6, s. 1081-1097Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The small ubiquitin-like modifier (SUMO) is as a regulator of many cellular functions by reversible conjugation to a broad number of substrates. Under endogenous or exogenous perturbations, the SUMO network becomes a fine sensor of stress conditions by alterations in the expression level of SUMO enzymes and consequently changing the status of SUMOylated proteins. The diaphragm is the major inspiratory muscle, which is continuously active under physiological conditions, but its structure and function is severely affected when passively displaced for long extents during mechanical ventilation (MV). An iatrogenic condition called Ventilator-Induced Diaphragm Dysfunction (VIDD) is a major cause of failure to wean patients from ventilator support but the molecular mechanisms underlying this dysfunction are not fully understood. Using a unique experimental Intensive Care Unit (ICU) rat model allowing long-term MV, diaphragm muscles were collected in rats control and exposed to controlled MV (CMV) for durations varying between 1 and 10 days. Endogenous SUMOylated diaphragm proteins were identified by mass spectrometry and validated with in vitro SUMOylation systems. Contractile, calcium regulator and mitochondrial proteins were of specific interest due to their putative involvement in VIDD. Differences were observed in the abundance of SUMOylated proteins between glycolytic and oxidative muscle fibers in control animals and high levels of SUMOylated proteins were present in all fibers during CMV. Finally, previously reported VIDD biomarkers and therapeutic targets were also identified in our datasets which may play an important role in response to muscle weakness seen in ICU patients. Data are available via ProteomeXchange with identifier PXD006085.

  • 32.
    Nielsen, Michael L.
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap, MMS, medicinsk masspektrometri.
    Savitski, Mikhail M.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap, MMS, medicinsk masspektrometri.
    Zubarev, Roman A.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap, MMS, medicinsk masspektrometri.
    Extent of modifications in human proteome samples and their effect on dynamic range of analysis in shotgun proteomics2006Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 5, nr 12, s. 2384-2391Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The complexity of the uman proteome, already enormous at the organism level, increases further in the course of the proteome analysis due to in vitro sample evolution. Most of in vitro alterations can also occur in vivo as post-translational modifications. These two types of modifications can only be distinguished a posteriori but not in the process of analysis, thus rendering necessary the analysis of every molecule in the sample. With the new software tool ModifiComb applied to MS/MS data, the extent of modifications was measured in tryptic mixtures representing the full proteome of human cells. The estimated level of 8-12 modified peptides per each unmodified tryptic peptide present at ≥1 % level is approaching one modification per amino acid on average. This is a higher modification rate than was previously thought, posing an additional challenge to analytical techniques. The solution to the problem is seen in improving sample preparation routines, introducing dynamic range-adjusted thresholds for database searches, using more specific MS/MS analysis using high mass accuracy and complementary fragmentation techniques, and revealing peptide families with identification of additional proteins only by unfamiliar peptides. Extensive protein separation prior to analysis reduces the requirements on speed and dynamic range of a tandem mass spectrometer and can be a viable alternative to the shotgun approach.

  • 33.
    Nielsen, Michael L
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Tekniska sektionen, Institutionen för teknikvetenskaper.
    Savitski, Mikhail M
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Tekniska sektionen, Institutionen för teknikvetenskaper.
    Zubarev, Roman A
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Tekniska sektionen, Institutionen för teknikvetenskaper, Jonfysik.
    Improving protein identification using complementary fragmentation techniques in Fourier transform mass spectrometry2005Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 4, nr 6, s. 835-845Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Identification of proteins by MS/MS is performed by matching experimental mass spectra against calculated spectra of all possible peptides in a protein data base. The search engine assigns each spectrum a score indicating how well the experimental data complies with the expected one; a higher score means increased confidence in the identification. One problem is the false-positive identifications, which arise from incomplete data as well as from the presence of misleading ions in experimental mass spectra due to gas-phase reactions, stray ions, contaminants, and electronic noise. We employed a novel technique of reduction of false positives that is based on a combined use of orthogonal fragmentation techniques electron capture dissociation (ECD) and collisionally activated dissociation (CAD). Since ECD and CAD exhibit many complementary properties, their combined use greatly increased the analysis specificity, which was further strengthened by the high mass accuracy (approximately 1 ppm) afforded by Fourier transform mass spectrometry. The utility of this approach is demonstrated on a whole cell lysate from Escherichia coli. Analysis was made using the data-dependent acquisition mode. Extraction of complementary sequence information was performed prior to data base search using in-house written software. Only masses involved in complementary pairs in the MS/MS spectrum from the same or orthogonal fragmentation techniques were submitted to the data base search. ECD/CAD identified twice as many proteins at a fixed statistically significant confidence level with on average a 64% higher Mascot score. The confidence in protein identification was hereby increased by more than 1 order of magnitude. The combined ECD/CAD searches were on average 20% faster than CAD-only searches. A specially developed test with scrambled MS/MS data revealed that the amount of false-positive identifications was dramatically reduced by the combined use of CAD and ECD.

  • 34. Noborn, Fredrik
    et al.
    Toledo, Alejandro Gomez
    Sihlbom, Carina
    Lengqvist, Johan
    Fries, Erik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kjellen, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Nilsson, Jonas
    Larson, Goran
    Identification of Chondroitin Sulfate Linkage Region Glycopeptides Reveals Prohormones as a Novel Class of Proteoglycans2015Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 14, nr 1, s. 41-49Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Vertebrates produce various chondroitin sulfate proteoglycans (CSPGs) that are important structural components of cartilage and other connective tissues. CSPGs also contribute to the regulation of more specialized processes such as neurogenesis and angiogenesis. Although many aspects of CSPGs have been studied extensively, little is known of where the CS chains are attached on the core proteins and so far, only a limited number of CSPGs have been identified. Obtaining global information on glycan structures and attachment sites would contribute to our understanding of the complex proteoglycan structures and may also assist in assigning CSPG specific functions. In the present work, we have developed a glycoproteomics approach that characterizes CS linkage regions, attachment sites, and identities of core proteins. CSPGs were enriched from human urine and cerebrospinal fluid samples by strong-anion-exchange chromatography, digested with chondroitinase ABC, a specific CSlyase used to reduce the CS chain lengths and subsequently analyzed by nLC-MS/MS with a novel glycopeptide search algorithm. The protocol enabled the identification of 13 novel CSPGs, in addition to 13 previously established CSPGs, demonstrating that this approach can be routinely used to characterize CSPGs in complex human samples. Surprisingly, five of the identified CSPGs are traditionally defined as prohormones (cholecystokinin, chromogranin A, neuropeptide W, secretogranin-1, and secretogranin-3), typically stored and secreted from granules of endocrine cells. We hypothesized that the CS side chain may influence the assembly and structural organization of secretory granules and applied surface plasmon resonance spectroscopy to show that CS actually promotes the assembly of chromogranin A core proteins in vitro. This activity required mild acidic pH and suggests that the CS-side chains may also influence the self-assembly of chromogranin A in vivo giving a possible explanation to previous observations that chromogranin A has an inherent property to assemble in the acidic milieu of secretory granules.

  • 35. Petruzziello, Filomena
    et al.
    Falasca, Sara
    Andrén, Per E.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap.
    Rainer, Gregor
    Zhang, Xiaozhe
    Chronic Nicotine Treatment Impacts the Regulation of Opioid and Non-opioid Peptides in the Rat Dorsal Striatum2013Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 12, nr 6, s. 1553-1562Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The chronic use of nicotine, the main psychoactive ingredient of tobacco smoking, alters diverse physiological processes and consequently generates physical dependence. To understand the impact of chronic nicotine on neuropeptides, which are potential molecules associated with dependence, we conducted qualitative and quantitative neuropeptidomics on the rat dorsal striatum, an important brain region implicated in the preoccupation/craving phase of drug dependence. We used extensive LC-FT-MS/MS analyses for neuropeptide identification and LC-FT-MS in conjunction with stable isotope addition for relative quantification. The treatment with chronic nicotine for 3 months led to moderate changes in the levels of endogenous dorsal striatum peptides. Five enkephalin opioid peptides were up-regulated, although no change was observed for dynorphin peptides. Specially, nicotine altered levels of nine non-opioid peptides derived from precursors, including somatostatin and cerebellin, which potentially modulate neurotransmitter release and energy metabolism. This broad but selective impact on the multiple peptidergic systems suggests that apart from the opioid peptides, several other peptidergic systems are involved in the preoccupation/craving phase of drug dependence. Our finding permits future evaluation of the neurochemical circuits modulated by chronic nicotine exposure and provides a number of novel molecules that could serve as potential therapeutic targets for treating drug dependence.

  • 36. Pivac, Nela
    et al.
    Knezevic, Ana
    Gornik, Olga
    Pucic, Maja
    Igl, Wilmar
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi.
    Peeters, Hilde
    Crepel, An
    Steyaert, Jean
    Novokmet, Mislav
    Redzic, Irma
    Nikolac, Matea
    Hercigonja, Vesna Novkovic
    Curkovic, Katarina Dodig
    Curkovic, Mario
    Nedic, Gordana
    Muck-Seler, Dorotea
    Borovecki, Fran
    Rudan, Igor
    Lauc, Gordan
    Human Plasma Glycome in Attention-Deficit Hyperactivity Disorder and Autism Spectrum Disorders2011Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 10, nr 1Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Over a half of all proteins are glycosylated, and their proper glycosylation is essential for normal function. Unfortunately, because of structural complexity of nonlinear branched glycans and the absence of genetic template for their synthesis, the knowledge about glycans is lagging significantly behind the knowledge about proteins or DNA. Using a recently developed quantitative high throughput glycan analysis method we quantified components of the plasma N-glycome in 99 children with attention-deficit hyperactivity disorder (ADHD), 81 child and 5 adults with autism spectrum disorder, and a total of 340 matching healthy controls. No changes in plasma glycome were found to associate with autism spectrum disorder, but several highly significant associations were observed with ADHD. Further structural analysis of plasma glycans revealed that ADHD is associated with increased antennary fucosylation of biantennary glycans and decreased levels of some complex glycans with three or four antennas. The design of this study prevented any functional conclusions about the observed associations, but specific differences in glycosylation appears to be strongly associated with ADHD and warrants further studies in this direction.

  • 37.
    Savitski, Mikhail M.
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Tekniska sektionen, Institutionen för teknikvetenskaper, Jonfysik. Jonfysik.
    Nielsen, Michael L.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Tekniska sektionen, Institutionen för teknikvetenskaper, Jonfysik. Jonfysik.
    Zubarev, Roman A.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Tekniska sektionen, Institutionen för teknikvetenskaper, Jonfysik. Jonfysik.
    ModifiComb: New proteomics tool for mapping substochiometric post-translational modifications, finding novel types of modifications and fingerprinting complex protein mixtures.2006Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 5, nr 5, s. 935-948Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A major challenge in proteomics is to fully identify and characterize the post-translational modification (PTM) patterns present at any given time in cells, tissues, and organisms. Here we present a fast and reliable method ("ModifiComb") for mapping hundreds types of PTMs at a time, including novel and unexpected PTMs. The high mass accuracy of Fourier transform mass spectrometry provides in many cases unique elemental composition of the PTM through the difference DeltaM between the molecular masses of the modified and unmodified peptides, whereas the retention time difference DeltaRT between their elution in reversed-phase liquid chromatography provides an additional dimension for PTM identification. Abundant sequence information obtained with complementary fragmentation techniques using ion-neutral collisions and electron capture often locates the modification to a single residue. The (DeltaM, DeltaRT) maps are representative of the proteome and its overall modification state and may be used for database-independent organism identification, comparative proteomic studies, and biomarker discovery. Examples of newly found modifications include +12.000 Da (+C atom) incorporation into proline residues of peptides from proline-rich proteins found in human saliva. This modification is hypothesized to increase the known activity of the peptide.

  • 38.
    Savitski, Mikhail M.
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Tekniska sektionen, Institutionen för teknikvetenskaper.
    Nielsen, Michael L.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Tekniska sektionen, Institutionen för teknikvetenskaper.
    Zubarev, Roman A.
    New data base-independent, sequence tag-based scoring of peptide MS/MS data validates Mowse scores, recovers below threshold data, singles out modified peptides, and assesses the quality of MS/MS techniques2005Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 4, nr 8, s. 1180-1188Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The Mascot score (M-score) is one of the conventional validity measures in data base identification of peptides and proteins by MS/MS data. Although tremendously useful, M-score has a number of limitations. For the same MS/MS data, M-score may change if the protein data base is expanded. A low M-value may not necessarily mean poor match but rather poor MS/MS quality. In addition M-score does not fully utilize the advantage of combined use of complementary fragmentation techniques collisionally activated dissociation (CAD) and electron capture dissociation (ECD). To address these issues, a new data base-independent scoring method (S-score) was designed that is based on the maximum length of the peptide sequence tag provided by the combined CAD and ECD data. The quality of MS/MS spectra assessed by S-score allows poor data (39% of all MS/MS spectra) to be filtered out before the data base search, speeding up the data analysis and eliminating a major source of false positive identifications. Spectra with below threshold M-scores (poor matches) but high S-scores are validated. Spectra with zero M-score (no data base match) but high S-score are classified as belonging to modified sequences. As an extension of S-score, an extremely reliable sequence tag was developed based on complementary fragments simultaneously appearing in CAD and ECD spectra. Comparison of this tag with the data base-derived sequence gives the most reliable peptide identification validation to date. The combined use of M- and S-scoring provides positive sequence identification from >25% of all MS/MS data, a 40% improvement over traditional M-scoring performed on the same Fourier transform MS instrumentation. The number of proteins reliably identified from Escherichia coli cell lysate hereby increased by 29% compared with the traditional M-score approach. Finally S-scoring provides a quantitative measure of the quality of fragmentation techniques such as the minimum abundance of the precursor ion, the MS/MS of which gives the threshold S-score value of 2.

  • 39.
    Scholz, Birger
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap.
    Sköld, Karl
    Kultima, Kim
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Klinisk farmakologi.
    Fernandez, Celine
    Waldemarson, Sofia
    Savitski, Mikhail M.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Söderquist, Marcus
    Borén, Mats
    Stella, Robert
    Andrén, Per
    Zubarev, Roman
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    James, Peter
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap.
    Impact of Temperature Dependent Sampling Procedures in Proteomics and Peptidomics: A Characterization of the Liver and Pancreas Post Mortem Degradome2011Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 10, nr 3, s. M900229MCP200-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Little is known about the nature of post mortem degradation of proteins and peptides on a global level, the so-called degradome. This is especially true for nonneural tissues. Degradome properties in relation to sampling procedures on different tissues are of great importance for the studies of, for instance, post translational modifications and/or the establishment of clinical biobanks. Here, snap freezing of fresh(< 2 min post mortem time) mouse liver and pancreas tissue is compared with rapid heat stabilization with regard to effects on the proteome (using two-dimensional differential in-gel electrophoresis) and peptidome (using label free liquid chromatography). We report several proteins and peptides that exhibit heightened degradation sensitivity, for instance superoxide dismutase in liver, and peptidyl-prolyl cis-trans isomerase and insulin C-peptides in pancreas. Tissue sampling based on snap freezing produces a greater amount of degradation products and lower levels of endogenous peptides than rapid heat stabilization. We also demonstrate that solely snap freezing related degradation can be attenuated by subsequent heat stabilization. We conclude that tissue sampling involving a rapid heat stabilization step is preferable to freezing with regard to proteomic and peptidomic sample quality.

  • 40. Schwenk, Jochen M.
    et al.
    Igel, Ulrika
    Neiman, Maja
    Langen, Hanno
    Becker, Charlotte
    Bjartell, Anders
    Pontén, Fredrik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi.
    Wiklund, Fredrik
    Grönberg, Henrik
    Nilsson, Peter
    Uhlén, Mathias
    Toward Next Generation Plasma Profiling via Heat-induced Epitope Retrieval and Array-based Assays2010Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 9, nr 11, s. 2497-2507Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 41.
    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 proteomics2007Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 6, nr 1, s. 125-132Artikel i tidskrift (Refereegranskat)
    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.

  • 42. Skoeld, Karl
    et al.
    Alm, Henrik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap.
    Scholz, Birger
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap.
    The Impact of Biosampling Procedures on Molecular Data Interpretation2013Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 12, nr 6, s. 1489-1501Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    The separation between biological and technical variation without extensive use of technical replicates is often challenging, particularly in the context of different forms of protein and peptide modifications. Biosampling procedures in the research laboratory are easier to conduct within a shorter time frame and under controlled conditions as compared with clinical sampling, with the latter often having issues of reproducibility. But is the research laboratory biosampling really less variable? Biosampling introduces within minutes rapid tissue-specific changes in the cellular microenvironment, thus inducing a range of different pathways associated with cell survival. Biosampling involves hypoxia and, depending on the circumstances, hypothermia, circumstances for which there are evolutionarily conserved defense strategies in the range of species and also are relevant for the range of biomedical conditions. It remains unclear to what extent such adaptive processes are reflected in different biosampling procedures or how important they are for the definition of sample quality. Lately, an increasing number of comparative studies on different biosampling approaches, postmortem effects and pre-sampling biological state, have investigated such immediate early biosampling effects. Commonalities between biosampling effects and a range of ischemia/reperfusion- and hypometabolism/anoxia-associated biological phenomena indicate that even small variations in post-sampling time intervals are likely to introduce a set of nonrandom and tissue-specific effects of experimental importance (both in vivo and in vitro). This review integrates the information provided by these comparative studies and discusses how an adaptive biological perspective in biosampling procedures may be relevant for sample quality issues.

  • 43. Stoop, Marcel P
    et al.
    Coulier, Leon
    Rosenling, Therese
    Shi, Shanna
    Smolinska, Agnieszka M
    Buydens, Lutgarde
    Ampt, Kirsten
    Stingl, Christoph
    Dane, Adrie
    Muilwijk, Bas
    Luitwieler, Ronald L
    Sillevis Smitt, Peter A E
    Hintzen, Rogier Q
    Bischoff, Rainer
    Wijmenga, Sybren S
    Hankemeier, Thomas
    van Gool, Alain J
    Luider, Theo M
    Quantitative proteomics and metabolomics analysis of normal human cerebrospinal fluid samples.2010Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 9, nr 9, s. 2063-75Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The analysis of cerebrospinal fluid (CSF) is used in biomarker discovery studies for various neurodegenerative central nervous system (CNS) disorders. However, little is known about variation of CSF proteins and metabolites between patients without neurological disorders. A baseline for a large number of CSF compounds appears to be lacking. To analyze the variation in CSF protein and metabolite abundances in a number of well-defined individual samples of patients undergoing routine, non-neurological surgical procedures, we determined the variation of various proteins and metabolites by multiple analytical platforms. A total of 126 common proteins were assessed for biological variations between individuals by ESI-Orbitrap. A large spread in inter-individual variation was observed (relative standard deviations [RSDs] ranged from 18 to 148%) for proteins with both high abundance and low abundance. Technical variation was between 15 and 30% for all 126 proteins. Metabolomics analysis was performed by means of GC-MS and nuclear magnetic resonance (NMR) imaging and amino acids were specifically analyzed by LC-MS/MS, resulting in the detection of more than 100 metabolites. The variation in the metabolome appears to be much more limited compared with the proteome: the observed RSDs ranged from 12 to 70%. Technical variation was less than 20% for almost all metabolites. Consequently, an understanding of the biological variation of proteins and metabolites in CSF of neurologically normal individuals appears to be essential for reliable interpretation of biomarker discovery studies for CNS disorders because such results may be influenced by natural inter-individual variations. Therefore, proteins and metabolites with high variation between individuals ought to be assessed with caution as candidate biomarkers because at least part of the difference observed between the diseased individuals and the controls will not be caused by the disease, but rather by the natural biological variation between individuals.

  • 44. Ståhl, Sara
    et al.
    Fung, Eva
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Adams, Christopher
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Lengqvist, Johan
    Mörk, Birgitta
    Stenerlöw, Bo
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för onkologi, radiologi och klinisk immunologi, Enheten för biomedicinsk strålningsvetenskap.
    Lewensohn, Rolf
    Lehtiö, Janne
    Zubarev, Roman
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Viktorsson, Kristina
    Proteomics and pathway analysis identifies JNK signaling as critical for high linear energy transfer radiation-induced apoptosis in non-small lung cancer cells2009Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 8, nr 5, s. 1117-1129Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    During the past decade, we have witnessed an explosive increase in generation of large proteomics data sets, not least in cancer research. There is a growing need to extract and correctly interpret information from such data sets to generate biologically relevant hypotheses. A pathway search engine (PSE) has recently been developed as a novel tool intended to meet these requirements. Ionizing radiation (IR) is an anticancer treatment modality that triggers multiple signal transduction networks. In this work, we show that high linear energy transfer (LET) IR induces apoptosis in a non-small cell lung cancer cell line, U-1810, whereas low LET IR does not. PSE was applied to study changes in pathway status between high and low LET IR to find pathway candidates of importance for high LET-induced apoptosis. Such pathways are potential clinical targets, and they were further validated in vitro. We used an unsupervised shotgun proteomics approach where high resolution mass spectrometry coupled to nanoflow liquid chromatography determined the identity and relative abundance of expressed proteins. Based on the proteomics data, PSE suggested the JNK pathway (p = 6.10(-6)) as a key event in response to high LET IR. In addition, the Fas pathway was found to be activated (p = 3.10(-5)) and the p38 pathway was found to be deactivated (p = 0.001) compared with untreated cells. Antibody-based analyses confirmed that high LET IR caused an increase in phosphorylation of JNK. Moreover pharmacological inhibition of JNK blocked high LET-induced apoptotic signaling. In contrast, neither an activation of p38 nor a role for p38 in high LET IR-induced apoptotic signaling was found. We conclude that, in contrast to conventional low LET IR, high LET IR can trigger activation of the JNK pathway, which in turn is critical for induction of apoptosis in these cells. Thus PSE predictions were largely confirmed, and PSE was proven to be a useful hypothesis-generating tool.

  • 45. Uhlen, Mathias
    et al.
    Bjorling, Erik
    Agaton, Charlotta
    Szigyarto, Cristina Al-Khalili
    Amini, Bahram
    Andersen, Elisabet
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Andersson, Ann-Catrin
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Angelidou, Pia
    Asplund, Anna
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Asplund, Caroline
    Berglund, Lisa
    Bergström, Kristina
    Brumer, Harry
    Cerjan, Dijana
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Ekstrom, Marica
    Elobeid, Adila
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Eriksson, Cecilia
    Fagerberg, Linn
    Falk, Ronny
    Fall, Jenny
    Forsberg, Mattias
    Björklund, Marcus Gry
    Gumbel, Kristoffer
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Halimi, Asif
    Hallin, Inga
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Hamsten, Carl
    Hansson, Marianne
    Hedhammar, My
    Hercules, Görel
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Kampf, Caroline
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Larsson, Karin
    Lindskog, Mats
    Lodewyckx, Wald
    Lund, Jan
    Lundeberg, Joakim
    Magnusson, Kristina
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Malm, Erik
    Nilsson, Peter
    Odling, Jenny
    Oksvold, Per
    Olsson, Ingmarie
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Oster, Emma
    Ottosson, Jenny
    Paavilainen, Linda
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Persson, Anja
    Rimini, Rebecca
    Rockberg, Johan
    Runeson, Marcus
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Sivertsson, Asa
    Sköllermo, Anna
    Steen, Johanna
    Stenvall, Maria
    Sterky, Fredrik
    Strömberg, Sara
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Sundberg, Mårten
    Department of Biotechnology, AlbaNova University Center, Royal Institute of Technology (KTH), Stockholm, Sweden.
    Tegel, Hanna
    Tourle, Samuel
    Wahlund, Eva
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Waldén, Annelie
    Wan, Jinghong
    Wernéus, Henrik
    Westberg, Joakim
    Wester, Kenneth
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Wrethagen, Ulla
    Xu, Lan Lan
    Hober, Sophia
    Pontén, Fredrik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    A human protein atlas for normal and cancer tissues based on antibody proteomics2005Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 4, nr 12, s. 1920-1932Artikel i tidskrift (Refereegranskat)
    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.

  • 46. Uhlén, Mathias
    et al.
    Oksvold, Per
    Algenäs, Cajsa
    Hamsten, Carl
    Fagerberg, Linn
    Klevebring, Daniel
    Lundberg, Emma
    Odeberg, Jacob
    Pontén, Fredrik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi.
    Kondo, Tadashi
    Sivertsson, Asa
    Antibody-based Protein Profiling of the Human Chromosome 212012Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 11, nr 3Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The Human Proteome Project has been proposed to create a knowledge-based resource based on a systematical mapping of all human proteins, chromosome by chromosome, in a gene-centric manner. With this background, we here describe the systematic analysis of chromosome 21 using an antibody-based approach for protein profiling using both confocal microscopy and immunohistochemistry, complemented with transcript profiling using next generation sequencing data. We also describe a new approach for protein isoform analysis using a combination of antibody-based probing and isoelectric focusing. The analysis has identified several genes on chromosome 21 with no previous evidence on the protein level, and the isoform analysis indicates that a large fraction of human proteins have multiple isoforms. A chromosome-wide matrix is presented with status for all chromosome 21 genes regarding subcellular localization, tissue distribution, and molecular characterization of the corresponding proteins. The path to generate a chromosome-specific resource, including integrated data from complementary assay platforms, such as mass spectrometry and gene tagging analysis, is discussed.

  • 47. van Duijn, Esther
    et al.
    Barbu, Ioana M.
    Barendreg, Arjan
    Jore, Matthijs M.
    Wiedenheft, Blake
    Lundgren, Magnus
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Westra, Edze R.
    Brouns, Stan J. J.
    Doudna, Jennifer A.
    van der Oost, John
    Heck, Albert J. R.
    Native Tandem and Ion Mobility Mass Spectrometry Highlight Structural and Modular Similarities in Clustered-Regularly-Interspaced Shot-Palindromic-Repeats (CRISPR)-associated Protein Complexes From Escherichia coli and Pseudomonas aeruginosa2012Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 11, nr 11, s. 1430-1441Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) immune system of bacteria and archaea provides acquired resistance against viruses and plasmids, by a strategy analogous to RNA-interference. Key components of the defense system are ribonucleoprotein complexes, the composition of which appears highly variable in different CRISPR/Cas subtypes. Previous studies combined mass spectrometry, electron microscopy, and small angle x-ray scattering to demonstrate that the E. coli Cascade complex (405 kDa) and the P. aeruginosa Csy-complex (350 kDa) are similar in that they share a central spiral-shaped hexameric structure, flanked by associating proteins and one CRISPR RNA. Recently, a cryo-electron microscopy structure of Cascade revealed that the CRISPR RNA molecule resides in a groove of the hexameric backbone. For both complexes we here describe the use of native mass spectrometry in combination with ion mobility mass spectrometry to assign a stable core surrounded by more loosely associated modules. Via computational modeling subcomplex structures were proposed that relate to the experimental IMMS data. Despite the absence of obvious sequence homology between several subunits, detailed analysis of sub-complexes strongly suggests analogy between subunits of the two complexes. Probing the specific association of E. coli Cascade/crRNA to its complementary DNA target reveals a conformational change. All together these findings provide relevant new information about the potential assembly process of the two CRISPR-associated complexes.

  • 48. Wiemhoefer, Anne
    et al.
    Stargardt, Anita
    van der Linden, Wouter A.
    Renner, Maria C.
    van Kesteren, Ronald E.
    Stap, Jan
    Raspe, Marcel A.
    Tomkinson, Birgitta
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kessels, Helmut W.
    Ovaa, Huib
    Overkleeft, Herman S.
    Florea, Bogdan
    Reits, Eric A.
    Tripeptidyl Peptidase II Mediates Levels of Nuclear Phosphorylated ERK1 and ERK22015Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 14, nr 8, s. 2177-2193Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Tripeptidyl peptidase II (TPP2) is a serine peptidase involved in various biological processes, including antigen processing, cell growth, DNA repair, and neuropeptide mediated signaling. The underlying mechanisms of how a peptidase can influence this multitude of processes still remain unknown. We identified rapid proteomic changes in neuroblastoma cells following selective TPP2 inhibition using the known reversible inhibitor butabindide, as well as a new, more potent, and irreversible peptide phosphonate inhibitor. Our data show that TPP2 inhibition indirectly but rapidly decreases the levels of active, di-phosphorylated extracellular signal-regulated kinase 1 (ERK1) and ERK2 in the nucleus, thereby down-regulating signal transduction downstream of growth factors and mitogenic stimuli. We conclude that TPP2 mediates many important cellular functions by controlling ERK1 and ERK2 phosphorylation. For instance, we show that TPP2 inhibition of neurons in the hippocampus leads to an excessive strengthening of synapses, indicating that TPP2 activity is crucial for normal brain function.

  • 49.
    Zieba, Agata
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Pardali, Katerina
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Söderberg, Ola
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Lindbom, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Nyström, Erik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Moustakas, Aristidis
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwiginstitutet för cancerforskning. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Heldin, Carl-Henrik
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwiginstitutet för cancerforskning. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Landegren, Ulf
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Intercellular variation in signaling through the TGF-β pathway and its relation to cell densityand cell cycle phase2012Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 11, nr 7, artikel-id M111.013482Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Fundamental open questions in signal transduction remain concerning the sequence and distribution of molecular signaling events among individual cells. In this work we have characterized the intercellular variability of transforming growth factor β-induced Smad interactions, providing essential information about TGF-β signaling and its dependence on the density of cell populations and the cell-cycle phase. By employing the recently developed in situ proximity ligation assay, we investigated the dynamics of interactions and modifications of Smad proteins and their partners under native and physiological conditions. We analyzed the kinetics of assembly of Smad complexes and the influence of cellular environment and relation to mitosis. We report rapid kinetics of formation of Smad complexes, including native Smad2-Smad3-Smad4 trimeric complexes, in a manner influenced by the rate of proteasomal degradation of these proteins, and we found a striking cell to cell variation of signaling complexes. The single-cell analysis of TGF-β signaling in genetically unmodified cells revealed previously unknown aspects of regulation of this pathway, and it provided a basis for analysis of these signaling events to diagnose pathological perturbations in patient samples, and to evaluate their susceptibility to drug treatment.

  • 50.
    Zubarev, Roman
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Tekniska sektionen, Institutionen för teknikvetenskaper, Jonfysik.
    Mann, M.
    On the proper use of mass accuracy in proteomics2007Ingår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 6, nr 3, s. 377-381Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Mass measurement is the main outcome of mass spectrometry-based proteomics yet the potential of recent advances in accurate mass measurements remains largely unexploited. There is not even a clear definition of mass accuracy in the proteomics literature, and we identify at least three uses of this term: anecdotal mass accuracy, statistical mass accuracy, and the maximum mass deviation (MMD) allowed in a database search. We suggest using the second of these terms as the generic one. To make the best use of the mass precision offered by modern instruments we propose a series of simple steps involving recalibration of the data on "internal standards" contained in every proteomics data set. Each data set should be accompanied by a plot of mass errors from which the appropriate MMD can be chosen. More advanced uses of high mass accuracy include an MMD that depends on the signal abundance of each peptide. Adapting search engines to high mass accuracy in the MS/MS data is also a high priority. Proper use of high mass accuracy data can make MS-based proteomics one of the most "digital" and accurate post-genomics disciplines.

1 - 50 av 50
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