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  • 1.
    Artemenko, Konstantin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Horakova, Jana
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Steinberger, Birgit
    Besenfelder, Urban
    Brem, Gottfried
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Mayrhofer, Corina
    A proteomic approach to monitor the dynamic response of the female oviductal epithelial cell surface to male gametes2015In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 113, p. 1-14Article in journal (Refereed)
    Abstract [en]

    UNLABELLED: Sophisticated strategies to analyze cell surface proteins are indispensable to study fundamental biological processes, such as the response of cells to environmental changes or cell-cell communication. Herein, we describe a refined mass spectrometry-based approach for the specific characterization and quantitation of cell surface proteins expressed in the female reproductive tract. The strategy is based on in situ biotinylation of rabbit oviducts, affinity enrichment of surface exposed biotin tagged proteins and dimethyl labeling of the obtained tryptic peptides followed by LC-MS/MS analysis. This approach proved to be sensitive enough to analyze small sample amounts (<1mug) and allowed further to trace the dynamic composition of the surface proteome of the oviductal epithelium in response to male gametes. The relative protein expression ratios of 175 proteins were quantified. Thirty-one of them were found to be altered over time, namely immediately, 1h and 2h after insemination compared to the time-matched control groups. Functional analysis demonstrated that structural reorganization of the oviductal epithelial cell surface was involved in the early response of the female organ to semen. In summary, this study outlines a workflow that is capable to monitor alterations in the female oviduct that are related to key reproductive processes in vivo. BIOLOGICAL SIGNIFICANCE: The proper interaction between the female reproductive tract, in particular, the oviduct and the male gametes, is fundamental to fertilization and embryonic development under physiological conditions. Thereby the oviductal epithelial cell surface proteins play an important role. Besides their direct interaction with male gametes, these molecules participate in signal transduction and, thus, are involved in the mandatory cellular response of the oviductal epithelium. In this study we present a refined LC-MS/MS based workflow that is capable to quantitatively analyze the expression of oviductal epithelial cell surface proteins in response to insemination in vivo. A special focus was on the very early interaction between the female organ and the male gametes. At first, this study clearly revealed an immediate response of the surface proteome to semen, which was modulated over time. The described methodology can be applied for studies of further distinct biological events in the oviduct and therefore contribute to a deeper insight into the formation of new life.

  • 2.
    Bayram, Helen L.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics. Univ Liverpool, Inst Integrat Biol, Mammalian Behav & Evolut Grp, Leahurst Campus, Neston CH64 7TE, England..
    Claydon, Amy J.
    Univ Liverpool, Inst Integrat Biol, Ctr Proteome Res, Liverpool L69 7ZB, Merseyside, England..
    Brownridge, Philip J.
    Univ Liverpool, Inst Integrat Biol, Ctr Proteome Res, Liverpool L69 7ZB, Merseyside, England..
    Hurst, Jane L.
    Univ Liverpool, Inst Integrat Biol, Mammalian Behav & Evolut Grp, Leahurst Campus, Neston CH64 7TE, England..
    Mileham, Alan
    Genus Plc, 1525 River Rd, De Forest, WI 53532 USA..
    Stockley, Paula
    Univ Liverpool, Inst Integrat Biol, Mammalian Behav & Evolut Grp, Leahurst Campus, Neston CH64 7TE, England..
    Beynon, Robert J.
    Univ Liverpool, Inst Integrat Biol, Ctr Proteome Res, Liverpool L69 7ZB, Merseyside, England..
    Hammond, Dean E.
    Univ Liverpool, Inst Translat Med, Cellular & Mol Physiol, Liverpool L69 3BX, Merseyside, England..
    Cross-species proteomics in analysis of mammalian sperm proteins2016In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 135, p. 38-50Article in journal (Refereed)
    Abstract [en]

    Many proteomics studies are conducted in model organisms for which fully annotated, detailed, high quality proteomes are available. By contrast, many studies in ecology and evolution are conducted in species which lack high quality proteome data, limiting the perceived value of a proteomic approach for protein discovery and quantification. This is particularly true of rapidly evolving proteins in the reproductive system, such as those that have an immune function or are under sexual selection, and can compromise the potential for cross-species proteomics to yield confident identification. In this investigation we analysed the sperm proteome, from a range of ungulates and rodents, and explored the potential of routine proteomic workflows to yield characterisation and quantification in non-model organisms. We report that database searching is robust to cross-species matching for a mammalian core sperm proteome, comprising 623 proteins that were common to most of the 19 species studied here, suggesting that these proteins are likely to be present and identifiable across many mammalian sperm. Further, label-free quantification reveals a consistent pattern of expression level. Functional analysis of this core proteome suggests consistency with previous studies limited to model organisms and has value as a quantitative reference for analysis of species-specific protein characterisation.

    Significance: From analysis of the sperm proteome for diverse species (rodents and ungulates) using LC-MS/MS workflows and standard data processing, we show that it is feasible to obtain cross-species matches for a large number of proteins that can be filtered stringently to yield a highly expressed mammalian sperm core proteome, for which label-free quantitative data are also used to inform protein function and abundance.

  • 3. Bespyatykh, Julia
    et al.
    Smolyakov, Alexander
    Guliaev, Andrei
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Shitikov, Egor
    Arapidi, Georgij
    Butenko, Ivan
    Dogonadze, Marine
    Manicheva, Olga
    Ilina, Elena
    Zgoda, Victor
    Govorun, Vadim
    Proteogenomic analysis of Mycobacterium tuberculosis Beijing B0/W148 cluster strains2019In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 192, p. 18-26Article in journal (Refereed)
    Abstract [en]

    Nowadays proteomics is one of the major instruments for editing and correcting annotation of genomic information. The correct genome annotation is necessary for omics studies of clinically relevant pathogens like Mycobacterium tuberculosis as well as for the progress in drug design and in silico biology. Here, we focused on the proteogenomic analysis of W-148 strain belonging to the Beijing B0/W148 cluster. This cluster, also known as a "successful" clone possesses unique pathogenic properties and has a unique genome organization. Taking into account high similarity of cluster strains at the genomic level we analyzed MS/MS dataset obtained for 63 clinical isolates of Beijing B0/W148. Based on H37Rv and W-148 annotations we identified 2546 proteins representing more than 60% of total proteome. A set of peptides (n = 404) specific for W-148 was found when compared with H37Rv. Start sites for 32 genes were corrected based on the combination of LC-MS/MS proteomic data with genomic six-frame translation. Additionally, we have shown the presence of peptides related to 10 genes earlier known as "pseudogenes". SIGNIFICANCE: Mycobacterium tuberculosis is one of the most dangerous pathogens. Phylogenetically, it may be divided into major lineages and among them, lineage 2 (predominantly Beijing genotype) one of the most successful lineages with an increasing prevalence in the global population. At the same time, strains of the Beijing B0/W148 cluster, a "successful" clone of Mycobacterium tuberculosis possess even more interesting features. Only one complete genome of this cluster, W-148, present in the NCBI database (CP012090.1) and it demonstrates a number of significant differences from the well-known reference genome H37Rv. For the W-148 strain many genes are annotated as "pseudo" and no attempts were made to correct this. Thereby, in this study, we have conducted a proteomic analysis of the cluster strains and corrected current genome annotation. We hope that the data obtained will help to increase the quality of identifications in proteomic and transcriptomic analysis of M. tuberculosis Beijing B0/W148 cluster strain in subsequent studies.

  • 4. Bozaykut, Perinur
    et al.
    Sozen, Erdi
    Kaga, Elif
    Ece, Asli
    Ozaltin, Esra
    Ek, Bo
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Ozer, Nesrin Kartal
    Grune, Tilman
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Karademir, Betul
    The role of heat stress on the age related protein carbonylation2013In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 89, p. 238-254Article in journal (Refereed)
    Abstract [en]

    Since the proteins are involved in many physiological processes in the organisms, modifications of proteins have important outcomes. Protein modifications are classified in several ways and oxidative stress related ones take a wide place. Aging is characterized by the accumulation of oxidized proteins and decreased degradation of these proteins. On the other hand protein turnover is an important regulatory mechanism for the control of protein homeostasis. Heat shock proteins are a highly conserved family of proteins in the various cells and organisms whose expressions are highly inducible during stress conditions. These proteins participate in protein assembly, trafficking, degradation and therefore play important role in protein turnover. Although the entire functions of each heat shock protein are still not completely investigated, these proteins have been implicated in the processes of protection and repair of stress-induced protein damage. This study has focused on the heat stress related carbonylated proteins, as a marker of oxidative protein modification, in young and senescent fibroblasts. The results are discussed with reference to potential involvement of induced heat shock proteins. This article is part of a Special Issue entitled: Protein Modifications. Biological significance Age-related protein modifications, especially protein carbonylation take a wide place in the literature. In this direction, to highlight the role of heat shock proteins in the oxidative modifications may bring a new aspect to the literature. On the other hand, identified carbonylated proteins in this study confirm the importance of folding process in the mitochondria which will be further analyzed in detail.

  • 5. de Wit, Meike
    et al.
    Kant, Huub
    Piersma, Sander R.
    Pham, Thang V.
    Mongera, Sandra
    van Berkel, Maaike P. A.
    Boven, Epie
    Pontén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Meijer, Gerrit A.
    Jimenez, Connie R.
    Fijneman, Remond J. A.
    Colorectal cancer candidate biomarkers identified by tissue secretome proteome profiling2014In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 99, p. 26-39Article in journal (Refereed)
    Abstract [en]

    Colorectal cancer (CRC) is a major health problem. Biomarkers associated with molecular changes in cancer cells can aid early detection, diagnosis, prognosis, therapy selection, and disease monitoring. Tumor tissue secretomes are a rich source of candidate biomarkers. To identify CRC protein biomarkers, secretomes of four pairs of human CRC tissue and patient-matched normal colon tissue samples, and secretomes of five CRC cell lines were analyzed by GeLC-MS/MS. Subsequent data analysis was based on label-free spectral counting, Ingenuity Pathway Analysis, Secretome/SignalP, STRING and Cytoscape, resulting in 2703 protein identifications in the tissue secretomes, of which 409 proteins were significantly more present in CRC samples than in controls. Biomarker selection of 76 candidates was based on consistent and abundant over-representation in cancer-compared to control-secretomes, and presumed neoplastic origin. Overlap analysis with previously obtained datasets revealed 21 biomarkers suited for early detection of CRC. Immunohistochemistry confirmed overexpression in CRC of one candidate marker (MCM5). In conclusion, a human reference dataset of 76 candidate biomarkers was identified for which we illustrate that combination with existing pre-clinical datasets allows pre-selection of biomarkers for blood- or stool-based assays to support clinical management of CRC. Further dedicated validation studies are required to demonstrate their clinical applicability. Biological significance Tissue secretome proteomes are a rich source of candidate biomarkers. Several secretome proteome datasets have been obtained from pre-clinical in vitro and in vivo colorectal cancer (CRC) model systems, yielding promising CRC biomarkers obtained under well-defined experimentally controlled conditions. However, which of these biomarker proteins are actually secreted by human CRC samples was not known. To our knowledge, this is the first study that directly compares secretome proteomes from clinically relevant human CRC tissues to patient-matched normal colon tissues. We identified 76 human CRC protein biomarkers that may facilitate blood-based or stool-based assay development to support clinical management of CRC. Overlap analysis with datasets from well-defined pre-clinical studies helps to determine what clinical application suits these human CRC biomarkers best, i.e. early detection, diagnosis, prognosis, therapy selection, and/or disease monitoring of CRC. This is demonstrated for a CRC mouse model dataset, revealing 21 human CRC biomarkers suited for early detection of CRC.

  • 6.
    Elf, Kristin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Neurology.
    Shevchenko, Ganna
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Nygren, Ingela
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Neurology.
    Larsson, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Clinical Neurophysiology.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Askmark, Håkan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Neurology.
    Artemenko, Konstantin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Alterations in muscle proteome of patients diagnosed with amyotrophic lateral sclerosis2014In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 108, p. 55-64Article in journal (Refereed)
    Abstract [en]

    Amyotrophic lateral sclerosis (ALS) is a motor neuron disease characterized by progressive muscle paralysis. Currently clinical tools for ALS diagnostics do not perform well enough and their improvement is needed. The objective of this study was to identify specific protein alterations related to the development of ALS using tiny muscle biopsies. We applied a shotgun proteomics and quantitative dimethyl labeling in order to analyze the global changes in human skeletal muscle proteome of ALS versus healthy subjects for the first time. 235 proteins were quantified and 11 proteins were found significantly regulated in ALS muscles. These proteins are involved in muscle development and contraction, metabolic processes, enzyme activity, regulation of apoptosis and transport activity. In order to eliminate a risk to confuse ALS with other denervations, muscle biopsies of patients with postpolio syndrome and Charcot Marie Tooth disease (negative controls) were compared to those of ALS and controls. Only few proteins significantly regulated in ALS patients compared to controls were affected differently in negative controls. These proteins (BTB and kelch domain-containing protein 10, myosin light chain 3, glycogen debranching enzyme, transitional endoplasmic reticulum ATPase), individually or as a panel, could be selected for estimation of ALS diagnosis and development. Biological significance ALS is a devastating neurodegenerative disease, and luckily, very rare: only one to two people out of 100,000 develop ALS yearly. This fact, however, makes studies of ALS very challenging since it is very difficult to collect the representative set of clinical samples and this may take up to several years. In this study we collected the muscle biopsies from 12 ALS patients and compared the ALS muscle proteome against the one from control subjects. We suggested the efficient method for such comprehensive quantitative analysis by LC-MS and performed it for the first time using human ALS material. This gel- and antibody-free method can be widely applied for muscle proteome studies and has been used by us for revealing of the specific protein alterations associated with ALS.

  • 7.
    Emami Khoonsari, Payam
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Musunri, Sravani
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Herman, Stephanie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Svensson, Camilla I
    Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden..
    Tanum, Lars
    Department of R&D in Mental Health, Akershus University Hospital, Lørenskog, Norway..
    Gordh, Torsten
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Anaesthesiology and Intensive Care.
    Kultima, Kim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Systematic Analysis of the Cerebrospinal Fluid Proteome of Fibromyalgia patients2019In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, p. 35-43Article in journal (Refereed)
    Abstract [en]

    Fibromyalgia (FM) is a syndrome characterized by widespread muscular pain, fatigue and functional symptoms, which is known to be difficult to diagnose as the various symptoms overlap with many other conditions. Currently, there are no biomarkers for FM, and the diagnosis is made subjectively by the clinicians. We have performed shotgun proteomics on cerebrospinal fluid (CSF) from FM patients and non-pain controls to find potential biomarker candidates for this syndrome. Based on our multivariate and univariate analyses, we found that the relative differences in the CSF proteome between FM patients and controls were moderate. Four proteins, important to discriminate FM patients from non-pain controls, were found: Apolipoprotein C-III, Galectin-3-binding protein, Malate dehydrogenase cytoplasmic and the neuropeptide precursor protein ProSAAS. These proteins are involved in lipoprotein lipase (LPL) activity, inflammatory signaling, energy metabolism and neuropeptide signaling.

  • 8.
    Emami Khoonsari, Payam
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry. Karolinska Inst, Dept Physiol & Pharmacol, Stockholm, Sweden.
    Ossipova, Elena
    Karolinska Univ Hosp, Karolinska Inst, Rheumatol Clin, Unit Rheumatol,Dept Med, Stockholm, Sweden.
    Lengqvist, Johan
    Karolinska Univ Hosp, Karolinska Inst, Rheumatol Clin, Unit Rheumatol,Dept Med, Stockholm, Sweden.
    Svensson, Camilla, I
    Karolinska Inst, Dept Physiol & Pharmacol, Stockholm, Sweden.
    Kosek, Eva
    Karolinska Inst, Dept Clin Neurosci, Stockholm, Sweden.
    Kadetoff, Diana
    Karolinska Inst, Dept Clin Neurosci, Stockholm, Sweden.
    Jakobsson, Per-Johan
    Karolinska Univ Hosp, Karolinska Inst, Rheumatol Clin, Unit Rheumatol,Dept Med, Stockholm, Sweden.
    Kultima, Kim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Lampa, Jon
    Karolinska Univ Hosp, Karolinska Inst, Rheumatol Clin, Unit Rheumatol,Dept Med, Stockholm, Sweden.
    The human CSF pain proteome2019In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 190, p. 67-76Article in journal (Refereed)
    Abstract [en]

    Chronic pain represents one of the major medical challenges in the 21st century, affecting > 1.5 billion of the world population. Overlapping and heterogenous symptoms of various chronic pain conditions complicate their diagnosis, emphasizing the need for more specific biomarkers to improve the diagnosis and understand the disease mechanisms. We have here investigated proteins found in human CSF with respect to known "pain" genes and in a cohort of patients with dysfunctional pain (fibromyalgia, FM), inflammatory pain (rheumatoid arthritis patients, RA) and non-pain controls utilized semi-quantitative proteomics using mass spectrometry (MS) to explore quantitative differences between these cohorts of patients. We found that "pain proteins" detected in CSF using MS are typically related to synaptic transmission, inflammatory responses, neuropeptide signaling- and hormonal activity. In addition, we found ten proteins potentially associated with chronic pain in FM and RA: neural cell adhesion molecule L1, complement C4-A, lysozyme C, receptor-type tyrosine-protein phosphatase zeta, apolipoprotein D, alpha-1-antichymotrypsin, granulins, calcium/calmodulin-dependent protein kinase type II subunit alpha, mast/stem cell growth factor receptor Kit, prolow-density lipoprotein receptor-related protein 1. These proteins might be of importance for understanding the mechanisms of dysfunctional/inflammatory chronic pain and also for use as potential biomarkers. Significance: Chronic pain is a common disease and it poses a large burden on worldwide health. Fibromyalgia (FM) is a heterogeneous disease of unknown etiology characterized by chronic widespread pain (CWP). The diagnosis and treatment of FM is based on the analysis of clinical assessments and no measurable biomarkers are available. Cerebrospinal fluid (CSF) has been historically considered as a rich source of biomarkers for diseases of nervous system including chronic pain. Here, we explore CSF proteome of FM patients utilizing mass spectrometry based quantitative proteomics method combined with multivariate data analysis in order to monitor the dynamics of the CSF proteome. Our findings in this exploratory study support notable presence of pain related proteins in CSF yet with specific domains including inflammatory responses, neuropeptide signaling- and hormonal activity. We have investigated molecular functions of significantly altered proteins and demonstrate presence of 176 known pain related proteins in CSF. In addition, we found ten proteins potentially associated with pain in FM and RA: neural cell adhesion molecule L1, complement C4-A, lysozyme C, receptor-type tyrosine-protein phosphatase zeta, apolipoprotein D, alpha-1-antichymotrypsin, granulins, calcium/calmodulindependent protein kinase type II subunit alpha, mast/stem cell growth factor receptor Kit, prolow-density lipoprotein receptor-related protein 1. These proteins are novel in the context of FM but are known to be involved in pain mechanisms including inflammatory response and signal transduction. These results should be of clear significance and interest for researchers and clinicians working in the field of pain utilizing human CSF and MS based proteomics.

  • 9.
    Goodwin, Richard J. A.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Sample preparation for mass spectrometry imaging: Small mistakes can lead to big consequences2012In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 75, no 16, p. 4893-4911Article, review/survey (Refereed)
    Abstract [en]

    Mass spectrometry imaging (MSI) enables the direct analysis of molecules from the surface of a wide variety of samples, allowing the multiplex measurement of both abundance and distribution of small molecules, lipids, peptides and proteins. As the technology has been refined an increasing number of ionization methods and mass analyzers has been used that enable increased spatial and spectral resolution measurements to be made at an increased speed. Alongside the instrumentation improvements there has been optimization of sample preparation procedures that allow the highest quality data to be obtained, reproducibly, from an ever increasing diversity of samples. This review will consider the development and standardization of sample preparation methods applicable to MSI, describing the stages and procedures undertaken from the instance of sample collection, through storage, preparation and on through final processing prior to analysis. Recent technical advancements will be highlighted and areas where further experimentation and optimization may well be required will be described. All aspects of the sample preparation pipeline will be considered in detail, with examples from the literature used to emphasize why rigorous sample preparation for MSI is vital to achieve the most accurate, reproducible and validated MSI data possible. This article is part of a Special Issue entitled: Imaging Mass Spectrometry: A User's Guide to a New Technique for Biological and Biomedical Research.

  • 10.
    Goodwin, Richard J. A.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Nilsson, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Borg, Daniel
    Langridge-Smith, Pat R. R.
    Harrison, David J.
    Mackay, C. Logan
    Iverson, Suzanne L.
    Andrén, Per E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Conductive carbon tape used for support and mounting of both whole animal and fragile heat-treated tissue sections for MALDI MS imaging and quantitation2012In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 75, no 16, p. 4912-4920Article in journal (Refereed)
    Abstract [en]

    Analysis of whole animal tissue sections by MALDI MS imaging (MSI) requires effective sample collection and transfer methods to allow the highest quality of in situ analysis of small or hard to dissect tissues. We report on the use of double-sided adhesive conductive carbon tape during whole adult rat tissue sectioning of carboxymethyl cellulose (CMC) embedded animals, with samples mounted onto large format conductive glass and conductive plastic MALDI targets, enabling MSI analysis to be performed on both TOF and FT-ICR MALDI mass spectrometers. We show that mounting does not unduly affect small molecule MSI detection by analyzing tiotropium abundance and distribution in rat lung tissues, with direct on-tissue quantitation achieved. Significantly, we use the adhesive tape to provide support to embedded delicate heat-stabilized tissues, enabling sectioning and mounting to be performed that maintained tissue integrity on samples that had previously been impossible to adequately prepare section for MSI analysis. The mapping of larger peptidomic molecules was not hindered by tape mounting samples and we demonstrate this by mapping the distribution of PEP-19 in both native and heat-stabilized rat brains. Furthermore, we show that without heat stabilization PEP-19 degradation fragments can detected and identified directly by MALDI MSI analysis.

    This article is part of a Special Issue entitled: Imaging Mass Spectrometry: A User's Guide to a New Technique for Biological and Biomedical Research.

  • 11.
    Kalayou, Shewit
    et al.
    Norwegian Univ Life Sci, Sect Expt Biomed, Oslo, Norway.;Mekelle Univ, Coll Vet Med, Mekelle, Ethiopia..
    Granum, Cesilie
    Norwegian Univ Life Sci, Sect Expt Biomed, Oslo, Norway..
    Berntsen, Hanne Friis
    Norwegian Univ Life Sci, Sect Expt Biomed, Oslo, Norway..
    Groseth, Per Kristian
    Norwegian Univ Life Sci, Sect Expt Biomed, Oslo, Norway..
    Verhaegen, Steven
    Norwegian Univ Life Sci, Sect Expt Biomed, Oslo, Norway..
    Connolly, Lisa
    Queens Univ Belfast, Inst Global Food Secur, Sch Biol Sci, Belfast BT7 1NN, Antrim, North Ireland..
    Brandt, Ingvar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Environmental toxicology.
    de Souza, Gustavo Antonio
    Univ Oslo, Oslo Univ Hosp, Dept Immunol, Rikshosp, POB 4950, N-0424 Oslo, Norway..
    Ropstad, Erik
    Norwegian Univ Life Sci, Sect Expt Biomed, Oslo, Norway..
    Label-free based quantitative proteomics analysis of primary neonatal porcine Leydig cells exposed to the persistent contaminant 3-methylsulfonyl-DDE2016In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 137, p. 68-82Article in journal (Refereed)
    Abstract [en]

    Evidence that persistent environmental pollutants may target the male reproductive system is increasing. The male reproductive system is regulated by secretion of testosterone by testicular Leydig cells, and perturbation of Leydig cell function may have ultimate consequences. 3-Methylsulfonyl-DDE (3-MeSO2-DDE) is a potent adrenal toxicants formed from the persistent insecticide DDT. Although studies have revealed the endocrine disruptive effect of 3-MeSO2-DDE, the underlying mechanisms at cellular level in steroidogenic Leydig cells remains to be established. The current study addresses the effect of 3-MeSO2-DDE on viability, hormone production and proteome response of primary neonatal porcine Leydig cells. The AlamarBlue (TM) assay was used to evaluate cell viability. Solid phase radioimmunoassay was used to measure concentration of hormones produced by both unstimulated and Luteinizing hormone (LH)-stimulated Leydig cells following 48 h exposure. Protein samples from Leydig cells exposed to a non-cytotoxic concentration of 3-MeSO2-DDE (10 mu M) were subjected to nano-LC-MS/MS and analyzed on a Q Exactive mass spectrometer and quantified using label-free quantitative algorithm. Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA) were carried out for functional annotation and identification of protein interaction networks. 3-MeSO2-DDE regulated Leydig cell steroidogenesis differentially depending on cell culture condition. Whereas its effect on testosterone secretion at basal condition was stimulatory, the effect on LH-stimulated cells was inhibitory. From triplicate experiments, a total of 6804 proteins were identified in which the abundance of 86 proteins in unstimulated Leydig cells and 145 proteins in LH-stimulated Leydig cells was found to be significantly regulated in response to 3-MeSO2-DDE exposure. These proteins not only are the first reported in relation to 3-MeSO2-DDE exposure, but also display small number of proteins shared between culture conditions, suggesting the action of 3-MeSO2-DDE on several targeted pathways, including mitochondrial dysfunction, oxidative phosphorylation, EIF2-signaling, and glutathione-mediated detoxification. Further identification and characterization of these proteins and pathways may build our understanding to the molecular basis of 3-MeSO2-DDE induced endocrine disruption in Leydig cells.

  • 12.
    Kjellander, Marcus
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Billinger, Erika
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Ramachandraiah, Harisha
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Boman, Mats
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Inorganic Chemistry.
    Bergström Lind, Sara
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Johansson, Gunnar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    A flow-through nanoporous alumina trypsin bioreactor for mass spectrometry peptide fingerprinting2018In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 172, p. 165-172Article in journal (Refereed)
    Abstract [en]

    Mass spectrometry-based proteomics benefits from efficient digestion of protein samples. In this study, trypsinwas immobilized on nanoporous anodized alumina membranes to create an enzyme reactor suitable for peptidemassfingerprinting. The membranes were derivatized with 3-aminopropyltriethoxysilane and the amino groupswere activated with carbonyldiimidazole to allow coupling of porcine trypsin viaε-amino groups. The functionwas assessed using the artificial substrate Nα-Benzoyl-L-arginine 4-nitroanilide hydrochloride, bovine ribonu-clease A and a human plasma sample. A 10-membraneflow-through reactor was used for fragmentation and MSanalysis after a single pass of substrate both by collection of product and subsequent off-line analysis, and bycoupling on-line to the instrument. The peptide pattern allowed correct identification of the single target proteinin both cases, and of > 70 plasma proteins in single pass mode followed by LC-MS analysis. The reactor retained76% of the initial activity after 14 days of storage and repeated use at room temperature.

    Significance:This manuscript describes the design of a stable enzyme reactor that allows efficient and fast di-gestion with negligible leakage of enzyme and enzyme fragments. The high stability facilitates the use in anonline-setup with MS detection since it allows the processing of multiple samples within an extended period of time without replacement.

  • 13.
    Kultima, Kim
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Sköld, Karl
    Borén, Mats
    Biomarkers of disease and post-mortem changes: Heat stabilization, a necessary tool for measurement of protein regulation2011In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 75, no 1, p. 145-159Article, review/survey (Refereed)
    Abstract [en]

    This review focuses on post sampling changes and how the Stabilizor system has been used to control this natural biological process and potential implications on cancer-specific biomarkers due to post sampling changes. Tissue sampling is a major traumatic event that can have drastic effects within a very short timeframe at the molecular level [1] resulting in loss of sample quality due to post-mortem changes. A heat-stabilization technology, using the Stabilizor system, has been developed to quickly and permanently abolish the enzymatic activity that causes these changes post-sampling and so preserve sample quality. The Stabilizor system has been shown to give better sample quality when analyzing a variety of tissues in various proteomic workflows. In this paper we discuss the impact of using heat-stabilized tissue in different proteomic applications. Based on our observations regarding the overlap between commonly changing proteins and proteins found to change post-mortem we also highlight a group of proteins of particular interest in cancer studies.

  • 14.
    Källback, Patrik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Shariatgorji, Mohammadreza
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Nilsson, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Andrén, Per E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Novel mass spectrometry imaging software assisting labeled normalization and quantitation of drugs and neuropeptides directly in tissue sections2012In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 75, no 16, p. 4941-4951Article in journal (Refereed)
    Abstract [en]

    MALDI MS imaging has been extensively used to produce qualitative distribution maps of proteins, peptides, lipids, small molecule pharmaceuticals and their metabolites directly in biological tissue sections. There is growing demand to quantify the amount of target compounds in the tissue sections of different organs. We present a novel MS imaging software including protocol for the quantitation of drugs, and for the first time, an endogenous neuropeptide directly in tissue sections. After selecting regions of interest on the tissue section, data is read and processed by the software using several available methods for baseline corrections, subtractions, denoising, smoothing, recalibration and normalization. The concentrations of in vivo administered drugs or endogenous compounds are then determined semi-automatically using either external standard curves, or by using labeled compounds, i.e., isotope labeled analogs as standards. As model systems, we have quantified the distribution of imipramine and tiotropium in the brain and lung of dosed rats. Substance P was quantified in different mouse brain structures, which correlated well with previously reported peptide levels. Our approach facilitates quantitative data processing and labeled standards provide better reproducibility and may be considered as an efficient tool to quantify drugs and endogenous compounds in tissue regions of interest.

  • 15.
    Lindskog, Cecilia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Korsgren, Olle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Pontén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Eriksson, Jan W
    Department of Molecular and Clinical Medicine, Sahlgrenska University Hospital, Gothenburg, Sweden.
    Johansson, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Radiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Danielsson, Angelika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Radiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Novel pancreatic beta cell-specific proteins: Antibody-based proteomics for identification of new biomarker candidates2012In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 75, no 9, p. 2611-2620Article in journal (Refereed)
    Abstract [en]

    Beta cell-specific surface targets are required for non-invasive monitoring of beta cell mass, which could be used for evaluation of new diabetes treatments as well as to help unravel pathogenic mechanisms underlying beta cell dysfunction. By antibody-based proteomics, we have identified and explored a set of islet cell-specific proteins. A search algorithm in the Human Protein Atlas was set up for identification of islet-specific proteins that yielded 27 hits, of which twelve showed a clear membranous expression pattern or had predicted transmembrane regions. The specificity of the identified proteins was investigated by immunohistochemical staining of pancreas sections from diabetic and non-diabetic subjects. No expression of these antigens could be detected in the exocrine pancreas. Colocalization with insulin and glucagon was further determined by confocal microscopy using isolated human islets. All antibodies specifically stained human islets and colocalization analysis revealed that four proteins were exclusively expressed in beta cells. Importantly, these antibodies were negative in sections from subjects with long-standing type 1 diabetes. In the present study, we present four proteins; DGCR2, GBF1, GPR44 and SerpinB10, the expression of which has not previously been described in beta cells.

  • 16. Malm, Johan
    et al.
    Fehniger, Thomas E.
    Danmyr, Pia
    Vegvari, Akos
    Welinder, Charlotte
    Lindberg, Henrik
    Appelqvist, Roger
    Sjodin, Karin
    Wieslander, Elisabet
    Laurell, Thomas
    Hober, Sophia
    Berven, Frode S.
    Fenyoe, David
    Wang, Xiangdong
    Andrén, Per E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Edula, Goutham
    Carlsohn, Elisabet
    Fuentes, Manuel
    Nilsson, Carol L.
    Dahlback, Magnus
    Rezeli, Melinda
    Erlinge, David
    Marko-Varga, Gyorgy
    Developments in biobanking workflow standardization providing sample integrity and stability2013In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 95, p. 38-45Article, review/survey (Refereed)
    Abstract [en]

    Recommendations and outlines for standardization in biobanking processes are presented by a research team with long-term experience in clinical studies. These processes have important bearing on the use of samples in developing assays. These measurements are useful to document states of health and disease that are beneficial for academic research, commercial healthcare, drug development industry and government regulating agencies. There is a need for increasing awareness within proteomic and genomic communities regarding the basic concepts of collecting, storing and utilizing clinical samples. Quality control and sample suitability for analysis need to be documented and validated to ensure data integrity and establish contexts for interpretation of results. Standardized methods in proteomics and genomics are required to be practiced throughout the community allowing datasets to be comparable and shared for analysis. For example, sample processing of thousands of clinical samples, performed in 384 high-density sample tube systems in a fully automated workflow, preserves sample content and is presented showing validation criteria. Large studies will be accompanied by biological and molecular information with corresponding clinical records from patients and healthy donors. These developments position biobanks of human patient samples as an increasingly recognized major asset in disease research, future drug development and within patient care. Biological significance The current manuscript is of major relevance to the proteomic and genomic fields, as it outlines the standardization aspects of biobanking and the requirements that are needed to run future clinical studies that will benefit the patients where OMICS science will play a major role. A global view of the field is given where best practice and conventional acceptances are presented along with ongoing large-scale biobanking projects. The authors represent broadly stakeholders that cover the academic, pharma, biotech and healthcare fields with extensive experience and deliveries. This contribution will be a milestone paper to the proteomic and genomic scientists to present data in the future that will have impact to the life science area. This article is part of a Special Issue entitled: Standardization and Quality Control in Proteomics.

  • 17.
    Malmström, Lars
    et al.
    University of Zurich, S3IT.
    Bakochi, Anahita
    Lund University, Department of Clinical Sciences.
    Svensson, Gabriel
    Lund University, Department of Clinical Sciences.
    Kilsgård, Ola
    Lund University, Department of Clinical Sciences.
    Lantz, Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Petersson, Ann Cathrine
    Region Skåne, Department of Clinical Microbiology.
    Hauri, Simon
    Lund University, Department of Clinical Sciences.
    Karlsson, Christofer
    Lund University, Department of Clinical Sciences.
    Malmström, Johan
    Lund University, Department of Clinical Sciences.
    Quantitative proteogenomics of human pathogens using DIA-MS2015In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 129, no SI, p. 98-107Article in journal (Refereed)
    Abstract [en]

    The increasing number of bacterial genomes in combination with reproducible quantitative proteome measurements provides new opportunities to explore how genetic differences modulate proteome composition and virulence. It is challenging to combine genome and proteome data as the underlying genome influences the proteome. We present a strategy to facilitate the integration of genome data from several genetically similar bacterial strains with data-independent analysis mass spectrometry (DIA-MS) for rapid interrogation of the combined data sets. The strategy relies on the construction of a composite genome combining all genetic data in a compact format, which can accommodate the fusion with quantitative peptide and protein information determined via DIA-MS. We demonstrate the method by combining data sets from whole genome sequencing, shotgun MS and DIA-MS from 34 clinical isolates of Streptococcus pyogenes. The data structure allows for fast exploration of the data showing that undetected proteins are on average more amenable to amino acid substitution than expressed proteins. We identified several significantly differentially expressed proteins between invasive and non-invasive strains. The work underlines how integration of whole genome sequencing with accurately quantified proteomes can further advance the interpretation of the relationship between genomes, proteomes and virulence. This article is part of a Special Issue entitled: Computational Proteomics.

  • 18. McDonnell, L
    et al.
    Andren, Per E
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Corthals, GL
    Imaging mass spectrometry: a user’s guide to a new technique for biological and biomedical research2012In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 75, no 16, p. 4881-4882Article in journal (Refereed)
  • 19. McDonnell, Liam A.
    et al.
    Heeren, Ron M. A.
    Andrén, Per E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Stoeckli, Markus
    Corthals, Garry L.
    Going forward: Increasing the accessibility of imaging mass spectrometry2012In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 75, no 16, p. 5113-5121Article, review/survey (Refereed)
    Abstract [en]

    The driving force behind the high and increasing popularity of imaging mass spectrometry is its demonstrated potential for the determination of new diagnostic/prognostic biomarkers and its ability to simultaneously trace the distributions of pharmaceuticals and their metabolites in tissues without the need to develop expensive radioactively-labeled analogues. Both of these applications would benefit from standardized methods, for the development of novel MS-based molecular histology tests and governmental-approved MS-based assays for pharmaceutical development. In addition, the broader scientific community would benefit from the increased accessibility of the technique. Currently imaging MS studies are individual endeavors, utilizing the individual expertise and infrastructure of a single laboratory and their immediate collaborators. A wide array of tissue preparation, data acquisition and data analysis techniques has been developed but lacks an international collaborative structure and data sharing capabilities. Such a collaborative framework would enable methodological exchange and detailed comparisons of analytical capabilities, to explore synergies between the different methods and result in the development of robust standardized methods. Here we describe the activities of a new European imaging MS network that will explicitly compare and contrast existing methods to provide best practice guidelines for the entire healthcare research community. This article is part of a Special Issue entitled: Imaging Mass Spectrometry: A User's Guide to a New Technique for Biological and Biomedical Research.

  • 20.
    Rimini, Rebecca
    et al.
    KTH, Proteomik.
    Schwenk, Jochen M.
    KTH, Proteomik.
    Sundberg, Marten
    Sjöberg, Ronald
    Klevebring, Daniel
    Gry, Marcus
    KTH, Proteomik.
    Uhlén, Mathias
    KTH, Proteomik.
    Nilsson, Peter
    KTH, Proteomik.
    Validation of serum protein profiles by a dual antibody array approach2009In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 73, no 2, p. 252-266Article in journal (Refereed)
    Abstract [en]

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

  • 21.
    Steinberger, Birgit
    et al.
    Univ Bodenkultur Wien, Interuniv Dept Agrobiotechnol IFA Tulln, Inst Biotechnol Anim Prod, Tulln, Austria.;Univ Vet Med, Dept Biomed Sci, Inst Anim Breeding & Genet, Vienna, Austria..
    Yu, Hans
    Univ Bodenkultur Wien, Interuniv Dept Agrobiotechnol IFA Tulln, Inst Biotechnol Anim Prod, Tulln, Austria.;Univ Vet Med, Dept Biomed Sci, Inst Anim Breeding & Genet, Vienna, Austria..
    Brodmann, Theodor
    Univ Bodenkultur Wien, Interuniv Dept Agrobiotechnol IFA Tulln, Inst Biotechnol Anim Prod, Tulln, Austria..
    Milovanovic, Daniela
    Med Univ Vienna, Clin Inst Pathol, Vienna, Austria..
    Reichart, Ursula
    Univ Vet Med Vienna, VetCore Facil Res, Vienna, Austria.;Univ Vet Med, Dept Biomed Sci, Inst Anim Breeding & Genet, Vienna, Austria..
    Besenfelder, Urban
    Univ Vet Med, Reprod Ctr Wieselburg, Vienna, Austria..
    Artemenko, Konstantin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Razzazi-Fazeli, Ebrahim
    Univ Vet Med Vienna, VetCore Facil Res, Vienna, Austria..
    Brem, Gottfried
    Univ Bodenkultur Wien, Interuniv Dept Agrobiotechnol IFA Tulln, Inst Biotechnol Anim Prod, Tulln, Austria.;Univ Vet Med, Dept Biomed Sci, Inst Anim Breeding & Genet, Vienna, Austria..
    Mayrhofer, Corina
    Univ Bodenkultur Wien, Interuniv Dept Agrobiotechnol IFA Tulln, Inst Biotechnol Anim Prod, Tulln, Austria.;Univ Vet Med, Dept Biomed Sci, Inst Anim Breeding & Genet, Vienna, Austria..
    Semen modulated secretory activity of oviductal epithelial cells is linked to cellular proteostasis network remodeling: Proteomic insights into the early phase of interaction in the oviduct in vivo2017In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 163, p. 14-27Article in journal (Refereed)
    Abstract [en]

    The oviductal epithelium is crucial for the integrity of the female organ. Previously we got evidence that the surface proteome of oviductal epithelial cells (Oecs) is promptly altered in response to insemination and thus suggested that this early phase plays a notable regulatory role in maintaining cellular function. This study further aimed to assess the effect of semen on the cellular and molecular mechanisms in rabbit Oecs. A quantitative gel-based proteomic approach was applied to analyze changes at three time points (0 h, I h, 2 h) after intrauterine insemination (IUI) compared to time matched controls. Within two hours the abundance of 22 protein species was evidently altered in the intracellular fraction. Functional analysis revealed that the proteins were primarily involved in proteostasis as well as metabolic processes. The analysis of phosphoproteins specified a role of mitogen-activated protein kinase (MAPK) signaling molecules. Concurrently, semen increased oviduct specific glycoprotein (OVGP1) secretion. A correlation between OVGP1 abundance and microtubule-associated proteins 1A/1B-light chain 3 lipidation was observed. The localization and changes in abundance of selected proteins were corroborated by antibody-based methods. These results clearly show that the early phase of interaction acts as a trigger for cellular adaptation to meet an altered demand in the female organ. Significance: The oviductal epithelium and its secreted proteins exert a pivotal role in reproductive processes, including the final maturation of male gametes. Thereby, the regulation and subsequently the functionality of the oviductal epithelial cell layer are important factors for the establishment of the appropriate milieu in the female reproductive tract. Notably, male gametes themselves have been shown to be an extrinsic modulatory factor of the oviductal epithelium. Accordingly a comprehensive knowledge about the underlying cellular and molecular mechanisms in the epithelial cells is of interest, also with regard to in vitro purposes. So far, the role of the early phase of interaction in the female organ has not been considered in detail. To get a further insight into the underlying cellular and molecular mechanisms, herein we analyzed the effect of semen on oviductal epithelial cells (Oecs) on the intracellular proteome level within the first two hours after insemination. The present study revealed a directed response of Oecs in vivo and disclosed intracellular pathways that are affected by the interplay between semen and the female reproductive tract. The prompt adaptation of the secretory activity and remodeling of the oviductal epithelium was accompanied by the concerted alterations of protein species that are primarily involved in the maintenance of cellular homeostasis. Besides emphasizing the importance of the early interaction phase for subsequent reproductive processes, the gained knowledge might further be implemented for in vitro applications as well.

  • 22.
    Valdés, Alberto
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Ström Holst, Bodil
    Swedish Univ Agr Sci, Dept Clin Sci, Uppsala, Sweden.
    Lindersson, Sebastian
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Ramström, Margareta
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Development of MS-based methods for identification and quantification of proteins altered during early pregnancy in dogs2019In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 192, p. 223-232Article in journal (Refereed)
    Abstract [en]

    Increased knowledge on serum protein profiles during early pregnancy in dogs would be valuable for several reasons, including animal welfare. Inflammatory changes during this period have been described. Today, mass spectrometry (MS) is a well-established technique to perform unbiased qualitative and quantitative studies of proteins in body fluids regardless of species. In the present study, a shotgun proteomic analysis based on nano-liquid chromatography-MS was performed to identify proteins of altered abundance during canine pregnancy, and, thereafter, a targeted parallel reaction monitoring (PRM)-method was developed and applied to absolutely quantify the concentrations of a selection of these proteins. Among the 32 proteins found altered between pregnant and non-pregnant dogs in the initial analysis, 12 were selected based on their changes in concentration and known biological importance, and these were analyzed using the PRM method. The PRM method showed good linearity, repeatability and sensitivity, and confirmed the higher concentration of Fibrinogen A, protein S alpha and C-reactive protein at early time points in pregnant bitches. In conclusion, the combination of both methods allowed the identification of several altered proteins, and the quantification and description of the concentration patterns for a selection of them during the early stage of dog pregnancy.

    Significance: MS is a powerful technique that allows the investigation of protein variations in samples from different origin, such as serum from dogs. The application of a shotgun proteomic analysis as a screening method has revealed the alteration of several proteins after fifteen days of pregnancy in dogs. The complementary development of a PRM MS-based method for several of these proteins has enabled the absolute quantification of their concentrations at five different time points during early pregnancy. With the MS technique, a combination of proteins can be studied with lower limits of detection than with immunoassays. Care should be taken not to interpret the observed changes in pregnant dogs as signs of disease.

  • 23.
    Valdés, Ana Elisa
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Physiological Botany.
    Irar, Sami
    Majadad, Juan P.
    Rodriguez, Ana
    Fernandez, Belen
    Pages, Montserrat
    Drought tolerance acquisition in Eucalyptus globulus (Labill.): A research on plant morphology, physiology and proteomics2013In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 79, p. 263-276Article in journal (Refereed)
    Abstract [en]

    Plants perceiving drought stress activate multiple responses to synchronise developmental and molecular activities aimed at improving survival. In this study we attained a multidisciplinary approach to examine the interplay among plant morphology, physiology and proteomics for understanding the mechanisms underlying the adaptive response to drought stress. The stress-related phenotype, the differential expression of putative members of the LEA family of proteins, the seed proteomic profile, and the endogenous content of free and conjugated abscisic acid (ABA and ABAGE) were analysed in two Eucalyptus globulus provenances with contrasting drought tolerance. Differences in morphology were noticeable, drought-tolerant genotypes displaying smaller seeds with higher desiccation in the mature state and a more developed root system that was not reduced under water stress treatments. From physiological and molecular points of view, the endogenous contents of ABA and ABAGE were also higher in the tolerant provenance, as well as the accumulation of proteins involved in abiotic stress tolerance processes. In addition, evidence of two immunologically-related proteins to the maize RAB17 and RAB28 proteins is first reported in Eucalyptus, showing similarities between species. Our results show that E. globulus displays simultaneous adjustments for acquiring drought tolerance that are expressed at physiological, developmental and molecular levels.

  • 24.
    Wadsworth, Caroline
    et al.
    Univ Manchester, Manchester Inst Biotechnol, Manchester M1 7DN, Lancs, England..
    Procopio, Noemi
    Univ Manchester, Manchester Inst Biotechnol, Manchester M1 7DN, Lancs, England..
    Anderung, Cecilia
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Evolutionary Biology.
    Carretero, Jose-Miguel
    Univ Burgos, Lab Evoluc Humana, Edificio I D I Plaza Misael Banuelos S-N, Burgos 09001, Spain.;La Trobe Univ, Dept Archaeol & Hist, Melbourne, Vic 3086, Australia..
    Iriarte, Eneko
    Univ Burgos, Lab Evoluc Humana, Edificio I D I Plaza Misael Banuelos S-N, Burgos 09001, Spain..
    Valdiosera, Cristina
    La Trobe Univ, Dept Archaeol & Hist, Melbourne, Vic 3086, Australia.;Univ Complutense Madrid, Ctr Mixto, Inst Salud Carlos Evoluc & Comportamiento Humanos, Madrid 28029, Spain..
    Elburg, Rengert
    Archaeol Heritage Off Saxony, Zur Wetterwarte 7, D-01109 Dresden, Germany..
    Penkman, Kirsty
    Univ York, Dept Chem, York YO10 5DD, N Yorkshire, England..
    Buckley, Michael
    Univ Manchester, Manchester Inst Biotechnol, Manchester M1 7DN, Lancs, England..
    Comparing ancient DNA survival and proteome content in 69 archaeological cattle tooth and bone samples from multiple European sites2017In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 158, p. 1-8Article in journal (Refereed)
    Abstract [en]

    Ancient DNA (aDNA) is the most informative biomolecule extracted from skeletal remains at archaeological sites, but its survival is unpredictable and its extraction and analysis is time consuming, expensive and often fails. Several proposed methods for better understanding aDNA survival are based upon the characterisation of some aspect of protein survival, but these are typically non-specific; proteomic analyses may offer an attractive method for understanding preservation processes. In this study, in-depth proteomic (LC-Orbitrap-MS/MS) analyses were carried out on 69 archaeological bovine bone and dentine samples from multiple European archaeological sites and compared with mitochondrial aDNA and amino acid racemisation (AAR) data. Comparisons of these data, including estimations of the relative abundances for seven selected non -collagenous proteins, indicate that the survival of aDNA in bone or dentine may correlate with the survival of some proteins, and that proteome complexity is a more useful predictor of aDNA survival than protein abundance or AAR. The lack of a strong correlation between the recovery of aDNA and the proteome abundance may indicate that the survival of aDNA is more closely linked to its ability to associate with bone hydroxyapatite crystals rather than to associate with proteins. Significance: Ancient biomolecule survival remains poorly understood, even with great advancements in 'omics' technologies, both in genomics and proteomics. This study investigates the survival of ancient DNA in relation to that of proteins, taking into account proteome complexity and the relative protein abundances to improve our understanding of survival mechanisms. The results show that although protein abundance is not necessarily directly related to aDNA survival, proteome complexity appears to be.

  • 25.
    Wisniewski, Jacek R.
    et al.
    Max Planck Inst Biochem, Biochem Prote Grp, Dept Prote & Signal Transduct, Klopferspitz 18, D-82152 Martinsried, Germany..
    Vildhede, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Norén, Agneta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Upper Abdominal Surgery.
    Artursson, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.
    In-depth quantitative analysis and comparison of the human hepatocyte and hepatoma cell line HepG2 proteomes2016In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 136, p. 234-247Article in journal (Refereed)
    Abstract [en]

    Hepatocytes play a pivotal role in human homeostasis. They are essential in regulation of glucose and lipid levels in blood and play a central role in metabolism of amino acids, lipids, drugs and xenobiotic-compounds. In addition, hepatocytes produce a major portion of proteins circulating in the blood. Hepatocytes were isolated from liver tissue obtained from surgical resections. Proteins were extracted and processed using filter aided sample preparation protocol and were analyzed by LC-MS/MS using high accuracy mass spectrometry. Proteins were quantified by the 'Total Protein Approach' and 'Proteomic Ruler'. We report a comprehensive proteomic analysis of purified human hepatocytes and the human hepatoma cell line HepG2. The complete dataset comprises 9400 proteins and provides a comprehensive and quantitative depiction of the proteomes of hepatocytes and HepG2 cells at the protein titer and copy number dimensions. We describe basic cell organization and in detail energy metabolism pathways and metabolite transport. We provide quantitative insights into protein synthesis and drug and xenobiotics catabolism. Our data delineate differences between the native human hepatocytes and HepG2 cells by providing for the first time quantitative data at protein concentrations and copy numbers. 

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