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  • 1.
    Brishammar, Sture
    et al.
    MASE Laboratory, Uppsala, Sweden.
    Hanrieder, Jörg
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    TMV-particle borne enhancer of a tobacco RNA-replicase2012In: World Journal of Science and Technology, ISSN 2231-2587, Vol. 2, no 7, p. 4-7Article in journal (Refereed)
    Abstract [en]

    According to separation studies it has been evident that a tobacco RNA-replicase after TMV-infection consists of two parts. The larger part is host-directed and will be combined with a virus borne small protein which considerably enhances the RNA-replicase activity and is therefore named replicase enhancer, Ree. This compound was found at HPLC-separations of TMV-coat proteins, and was detected using polymerase assay with a radioactive nucleotide involved. Molecular weight has been determined by mass spectrometry: with FT ICR MS to get the size – 6 023.3 - and with MALDI TOF MS to obtain a sequence of the 54 amino acids involved. Presumably Ree is fixed to the TMV-RNA at infection. The enzyme seems to produce minus-strands of the virus RNA.

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  • 2.
    Brittebo, Eva
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Karlsson, Oskar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Andersson, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Berg, Anna-Lena
    Roman, Erika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Lindquist, Nils Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Hanrieder, Jörg
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Neurotoxin-induced fibril formation and protein changes in rodents2012In: Toxicology Letters, ISSN 0378-4274, E-ISSN 1879-3169, Vol. 211, no Suppl., p. S193-193Article in journal (Other academic)
  • 3.
    Ekegren, Titti
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Hanrieder, Jörg
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Aquilonius, Sten-Magnus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Neurology.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Focused proteomics in post-mortem human spinal cord2006In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 5, no 9, p. 2364-2371Article in journal (Refereed)
    Abstract [en]

    With a highly sensitive electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) system, proteins were identified in minimal amounts of spinal cord from patients with the neurodegenerative disease amyotrophic lateral sclerosis (ALS) and compared to proteins in spinal cord from control subjects. The results show 18 versus 16 significantly identified ( p < 0.05) proteins, respectively, all known to be found in the central nervous system. The most abundant protein in both groups was the glial fibrillary acidic protein, GFAP. Other proteins were, for example, hemoglobin alpha- and, chain, myelin basic protein, thioredoxin, R enolase, and cholin acetyltransferase. This study also includes the technique of laser microdissection in combination with pressure catapulting (LMPC) for the dissection of samples and specific neurons. Furthermore, complementary experiments with nanoLC-matrix assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS) confirmed the results of the ESI-FTICR MS screening and provided additional results of further identified proteins.

  • 4.
    Ekegren, Titti
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Hanrieder, Jörg
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Clinical perspectives of high-resolution mass spectrometry-based proteomics in neuroscience: Exemplified in amyotrophic lateral sclerosis biomarker discovery research2008In: Journal of Mass Spectrometry, ISSN 1076-5174, E-ISSN 1096-9888, Vol. 43, no 5, p. 559-571Article in journal (Refereed)
    Abstract [en]

    Biomarker discovery is a central application in today's proteomic research. There is an urgent need for valid biomarkers to improve diagnostic tools and treatment in many disorders, such as the rapidly progressing neurodegenerative disorder amyotrophic lateral sclerosis (ALS) that has a fatal outcome in about 3 years and yet no curative treatment. Screening for clinically relevant biomarkers puts high demands on high-throughput, rapid and precise proteomic techniques. There is a large variety in the methods of choice involving mainly gel-based approaches as well as chromatographic techniques for multi-dimensional protein and peptide separations followed by mass spectrometry (MS) analysis. This special feature article will discuss some important aspects of MS-based clinical proteomics and biomarker discovery in the field of neuro degenerative diseases and ALS research respectively, with the aim to provide a prospective view on current and future research aspects in the field. Furthermore, examples for application of high-resolution MS-based proteomic strategies for ALS biomarker discovery will be demonstrated with two studies previously reported by our group. These studies include among others, utilization of capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS) for advanced protein pattern classification in cerebrospinal fluid (CSF) samples of ALS patients as well as highly sensitive protein identification in minimal amounts of postmortem spinal cord tissue and laser micro-dissected motor neurons using FT-ICR-MS in conjunction with nanoflow LC coupled to matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (LC-MALDI-TOF-TOF-MS).

  • 5.
    Fedulova, Natalia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Hanrieder, Jörg
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Emrén, Lars O.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Expression and purification of catalytically active human PHD3 in Escherichia coli2007In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 54, no 1, p. 1-10Article in journal (Refereed)
    Abstract [en]

    Transcription factor HIF-1 is a key regulator in cellular adaptation to hypoxia. HIF prolyl hydroxylases (PHDs) control HIF-1 accumulation by hydroxylation dependent on molecular oxygen. Due to this regulation, PHDs have been pointed out as potential drug targets. We have purified catalytically active human PHD3 after heterologous expression in Escherichia coli. Histidine-tagged enzyme was isolated as monomer by immobilized Ni-affinity chromatography followed by gel filtration. Overexpression of bacterial chaperonins GroEL/ES at 30 °C substantially increased the yield of soluble PHD3. High concentrations of salt and reducing agent during purification prevented protein aggregation. The enzyme activity with peptide derived from HIF-1α was inhibited by Zn2+, desferrioxamine and imidazole. The hydroxylation activity was verified by mass spectrometry, and Pro567 in HIF-1α was discovered as a new site of hydroxylation.

  • 6. Fuevesi, J.
    et al.
    Danielsson, Rolf
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Bencsik, K.
    Hanrieder, Jörg
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Zsiros, V.
    Rajda, C.
    Hakansson, P.
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy.
    Vecsei, L.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Cerebrospinal fluid proteome analysis reveals differentially abundant proteins in multiple sclerosis2014In: Multiple Sclerosis Journal, ISSN 1352-4585, E-ISSN 1477-0970, Vol. 20, p. 180-181Article in journal (Refereed)
  • 7.
    Füvesi, Judit
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Hanrieder, Jörg
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Bencsik, Krisztina
    Department of Neurology, Albert Szent-Györgyi Clinical Center, University of Szeged, Hungary.
    Rajda, Cecilia
    Department of Neurology, Albert Szent-Györgyi Clinical Center, University of Szeged, Hungary AND Department of Clinical Neurophysiology, Danish Epilepsy Centre, Denmark.
    Kovács, S. Krisztián
    Department of Pathology, Albert Szent-Györgyi Clinical Center, University of Szeged, Hungary .
    Kaizer, László
    Department of Pathology, Albert Szent-Györgyi Clinical Center, University of Szeged, Hungary .
    Beniczky, Sándor
    Department of Neurology, Albert Szent-Györgyi Clinical Center, University of Szeged, Hungary AND Department of Clinical Neurophysiology, Danish Epilepsy Centre, Denmark.
    Vécsei, László
    Department of Neurology, Albert Szent-Györgyi Clinical Center, University of Szeged AND Neuroscience Research Group, Hungarian Academy of Sciences, University of Szeged, Hungary.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Proteomic Analysis of Cerebrospinal Fluid in a Fulminant Case of Multiple Sclerosis2012In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 13, no 6, p. 7676-7693Article in journal (Refereed)
    Abstract [en]

    Multiple Sclerosis (MS) is a chronic disease, but in rare fulminant cases rapid progression may lead to death shortly after diagnosis. Currently there is no diagnostic test to predict disease course. The aim of this study was to identify potential biomarkers/proteins related to rapid progression. We present the case history of a 15-year-old male MS patient. Cerebrospinal fluid (CSF) was taken at diagnosis and at the time of rapid progression leading to the patient’s death. Using isobaric tag labeling and nanoflow liquid chromatography in conjunction with matrix assisted laser desorption/ionization time of flight tandem mass spectrometry we quantitatively analyzed the protein content of two CSF samples from the patient with fulminant MS as well as one relapsing-remitting (RR) MS patient and one control headache patient, whose CSF analysis was normal. Seventy-eight proteins were identified and seven proteins were found to be more abundant in both fulminant MS samples but not in the RR MS sample compared to the control. These proteins are involved in the immune response, blood coagulation, cell proliferation and cell adhesion. In conclusion, in this pilot study we were able to show differences in the CSF proteome of a rapidly progressing MS patient compared to a more typical clinical form of MS and a control subject.

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  • 8.
    Hanrieder, Jörg
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Cerebrum Illuminans: Mass Spectrometric Analysis of Protein and Peptide Dynamics in Neurological Diseases2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The human brain (lat. cerebrum) is the most complex and heterogeneous organ in the human body. It is involved in a great number of body functions like movement, touch sensing, vision, hearing, smelling, hormone regulation and many more. In no other organ, the molecular communication mechanisms between different cells are so poorly understood. Due to the extensive diversity of processes that are controlled by the brain, diseases and injuries of the nervous system affect the human body significantly. Because of the immense complexity of the brain, the molecular mechanisms underlying the pathology of the diseases remain largely unknown.

    Hence, there is an urgent need for the development of new analytical strategies in order to investigate these conditions on a molecular level. Here, a central focus lies in the study of protein and peptide expression profiles, which can provide an insight in ongoing molecular mechanisms underlying the pathophysiology of the diseases. A powerful approach for studying proteins and peptide dynamics is mass spectrometry based proteomics, which is defined as the comprehensive study of all proteins expressed in a biological matrix at a certain point of time.

    The central objective of this thesis was to develop and employ different mass spectrometric techniques to study protein and peptide dynamics in the central nervous system in different neurological diseases. The individual studies comprise different aspects of proteome research. The first two studies included clinical proteomic applications for investigating protein dynamics in traumatic brain injury and amyotrophic lateral sclerosis. A further study was focused on method development for MS analysis of intact neural cells. The final three projects described in this thesis comprised MS based protein and peptide imaging in brain and spinal cord tissue samples. Here, the aim was to elucidate topological changes in protein expression in ALS as well as neuropeptide alterations in distinct brain structures in L-DOPA induced dyskinesia (LID) in Parkinson’s disease.

    List of papers
    1. Temporally resolved differential proteomic analysis of human ventricular CSF for monitoring traumatic brain injury biomarker candidates.
    Open this publication in new window or tab >>Temporally resolved differential proteomic analysis of human ventricular CSF for monitoring traumatic brain injury biomarker candidates.
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    2009 (English)In: Journal of Neuroscience Methods, ISSN 0165-0270, E-ISSN 1872-678X, Vol. 177, no 2, p. 469-478Article in journal (Refereed) Published
    Abstract [en]

    A shotgun proteomic approach based on nanoflow liquid chromatography (nanoLC) in conjunction with matrix assisted laser desorption/ionization time of flight tandem mass spectrometry (MALDI TOF MS/MS) was utilized to quantitatively analyze the protein content of consecutive ventricular cerebrospinal fluid (CSF) samples of severe traumatic brain injury (TBI) patients on an individual basis. CSF was acquired from the lateral ventricle 1–9 days after the TBI incident by canula drain to investigate temporally resolved protein changes in three patients that required intracranial pressure monitoring during neurointensive care. The samples were subjected to at once tryptic digestion followed by isobaric tag labeling before multiplexed peptide separation and MS analysis. By using this approach, we were able to follow characteristic changes in protein concentrations over time allowing new conclusions to be drawn about ongoing pathological processes during TBI. Certain suggested protein-biomarker candidates for TBI, like acute phase reactants (APRs), fibrinogens (FIB), cystatin C (CC) or more brain specific proteins like glial fibrillary acid protein (GFAP) and neuron-specific enolase (NSE) were found to be significantly up-regulated which is in strong consistence with previously reported results. This methodology appears to be a promising tool for studying candidate biomarkers of neurovascular and traumatic brain injuries in the neurointensive care setting.

    Keywords
    Traumatic brain injury (TBI), ventricular cerebrospinal fluid (CSF), shotgun proteomics, protein quantification, isobaric tag labeling, matrix assisted laser desorption/ionization, time of flight tandem mass spectrometry (MALDI TOF MS/MS)
    National Category
    Analytical Chemistry
    Research subject
    Analytical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-88519 (URN)10.1016/j.jneumeth.2008.10.038 (DOI)000263393300027 ()19263575 (PubMedID)
    Available from: 2009-02-03 Created: 2009-02-03 Last updated: 2022-01-28Bibliographically approved
    2. Focused proteomics in post-mortem human spinal cord
    Open this publication in new window or tab >>Focused proteomics in post-mortem human spinal cord
    2006 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 5, no 9, p. 2364-2371Article in journal (Refereed) Published
    Abstract [en]

    With a highly sensitive electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) system, proteins were identified in minimal amounts of spinal cord from patients with the neurodegenerative disease amyotrophic lateral sclerosis (ALS) and compared to proteins in spinal cord from control subjects. The results show 18 versus 16 significantly identified ( p < 0.05) proteins, respectively, all known to be found in the central nervous system. The most abundant protein in both groups was the glial fibrillary acidic protein, GFAP. Other proteins were, for example, hemoglobin alpha- and, chain, myelin basic protein, thioredoxin, R enolase, and cholin acetyltransferase. This study also includes the technique of laser microdissection in combination with pressure catapulting (LMPC) for the dissection of samples and specific neurons. Furthermore, complementary experiments with nanoLC-matrix assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS) confirmed the results of the ESI-FTICR MS screening and provided additional results of further identified proteins.

    Keywords
    proteomics, electrospray ionization - Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS), matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS), neurodegeneration, amyotrophic lateral sclerosis, spinal cord, laser microdissection with pressure catapulting (LMPC)
    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-82961 (URN)10.1021/pr060237f (DOI)000240200700033 ()16944948 (PubMedID)
    Available from: 2006-10-25 Created: 2006-10-25 Last updated: 2017-12-14Bibliographically approved
    3. MALDI mass spectrometry based molecular phenotyping of CNS glial cells for prediction in mammalian brain tissue
    Open this publication in new window or tab >>MALDI mass spectrometry based molecular phenotyping of CNS glial cells for prediction in mammalian brain tissue
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    2011 (English)In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 401, no 1, p. 135-147Article in journal (Refereed) Published
    Abstract [en]

    The development of powerful analytical techniques for specific molecular characterization of neural cell types is of central relevance in neuroscience research for elucidating cellular functions in the central nervous system (CNS). This study examines the use of differential protein expression profiling of mammalian neural cells using direct analysis by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). MALDI-MS analysis is rapid, sensitive, robust, and specific for large biomolecules in complex matrices. Here, we describe a newly developed and straightforward methodology for direct characterization of rodent CNS glial cells using MALDI-MS-based intact cell mass spectrometry (ICMS). This molecular phenotyping approach enables monitoring of cell growth stages, (stem) cell differentiation, as well as probing cellular responses towards different stimulations. Glial cells were separated into pure astroglial, microglial, and oligodendroglial cell cultures. The intact cell suspensions were then analyzed directly by MALDI-TOF-MS, resulting in characteristic mass spectra profiles that discriminated glial cell types using principal component analysis. Complementary proteomic experiments revealed the identity of these signature proteins that were predominantly expressed in the different glial cell types, including histone H4 for oligodendrocytes and S100-A10 for astrocytes. MALDI imaging MS was performed, and signature masses were employed as molecular tracers for prediction of oligodendroglial and astroglial localization in brain tissue. The different cell type specific protein distributions in tissue were validated using immunohistochemistry. ICMS of intact neuroglia is a simple and straightforward approach for characterization and discrimination of different cell types with molecular specificity.

    Keywords
    Intact cell mass spectrometry (ICMS), MALDITOF- MS, Imaging mass spectrometry (IMS), Glial cells
    National Category
    Analytical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-156047 (URN)10.1007/s00216-011-5043-y (DOI)000292156900012 ()21553124 (PubMedID)
    Available from: 2011-07-08 Created: 2011-07-08 Last updated: 2022-01-28Bibliographically approved
    4. MALDI Imaging of Post Mortem Spjnal Cord in Amyotrophic Lateral Sclerosis
    Open this publication in new window or tab >>MALDI Imaging of Post Mortem Spjnal Cord in Amyotrophic Lateral Sclerosis
    (English)Manuscript (preprint) (Other academic)
    Identifiers
    urn:nbn:se:uu:diva-132440 (URN)
    Available from: 2010-10-20 Created: 2010-10-20 Last updated: 2012-10-18
    5. Imaging mass spectrometry reveals association of nigral levels of dynorphin peptides and L-DOPA-induced dyskinesia in Parkinson’s disease
    Open this publication in new window or tab >>Imaging mass spectrometry reveals association of nigral levels of dynorphin peptides and L-DOPA-induced dyskinesia in Parkinson’s disease
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    (English)Article in journal (Refereed) Submitted
    Identifiers
    urn:nbn:se:uu:diva-132438 (URN)
    Available from: 2010-10-20 Created: 2010-10-20 Last updated: 2011-08-22Bibliographically approved
    6. Alterations of striatal dynorphin peptide levels associated with L-DOPA-induced dyskinesia elucidated by imaging mass spectrometry
    Open this publication in new window or tab >>Alterations of striatal dynorphin peptide levels associated with L-DOPA-induced dyskinesia elucidated by imaging mass spectrometry
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    2010 (English)Manuscript (preprint) (Other academic)
    Publisher
    p. 96
    Identifiers
    urn:nbn:se:uu:diva-132441 (URN)
    Available from: 2010-10-28 Created: 2010-10-20 Last updated: 2022-01-28
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    FULLTEXT01
  • 9.
    Hanrieder, Jörg
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Mass Spectrometry Based Protein Biomarker Discovery in Clinical Specimens2008Licentiate thesis, comprehensive summary (Other academic)
  • 10.
    Hanrieder, Jörg
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Karlsson, Anna C.K.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Cordeiro, Helena
    Eriksson Mammo, Sofie
    Fälth, Maria
    German Cancer Research Center.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Andersson, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Alterations of striatal dynorphin peptide levels associated with L-DOPA-induced dyskinesia elucidated by imaging mass spectrometry2010Manuscript (preprint) (Other academic)
  • 11.
    Hanrieder, Jörg
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Chalmers, Dept Chem & Biol Engn, Gothenburg, Sweden.
    Ljungdahl, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Andersson, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease2012In: Journal of Visualized Experiments, E-ISSN 1940-087X, Vol. 14, no 60, article id e3445Article in journal (Refereed)
    Abstract [en]

    MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples(2) (Fig.1). A 12 μm thin tissue section is covered with a MALDI matrix, which facilitates desorption and ionization of intact peptides and proteins that can be detected with a mass analyzer, typically using a MALDI TOF/TOF mass spectrometer. Generally hundreds of peaks can be assessed in a single rat brain tissue section. In contrast to commonly used imaging techniques, this approach does not require prior knowledge of the molecules of interest and allows for unsupervised and comprehensive analysis of multiple molecular species while maintaining high molecular specificity and sensitivity(2). Here we describe a MALDI IMS based approach for elucidating region-specific distribution profiles of neuropeptides in the rat brain of an animal model Parkinson's disease (PD). PD is a common neurodegenerative disease with a prevalence of 1% for people over 65 of age(3,4). The most common symptomatic treatment is based on dopamine replacement using L-DOPA(5). However this is accompanied by severe side effects including involuntary abnormal movements, termed L-DOPA-induced dyskinesias (LID)(1,3,6). One of the most prominent molecular change in LID is an upregulation of the opioid precursor prodynorphin mRNA(7). The dynorphin peptides modulate neurotransmission in brain areas that are essentially involved in movement control(7,8). However, to date the exact opioid peptides that originate from processing of the neuropeptide precursor have not been characterized. Therefore, we utilized MALDI IMS in an animal model of experimental Parkinson's disease and L-DOPA induced dyskinesia. MALDI imaging mass spectrometry proved to be particularly advantageous with respect to neuropeptide characterization, since commonly used antibody based approaches targets known peptide sequences and previously observed post-translational modifications. By contrast MALDI IMS can unravel novel peptide processing products and thus reveal new molecular mechanisms of neuropeptide modulation of neuronal transmission. While the absolute amount of neuropeptides cannot be determined by MALDI IMS, the relative abundance of peptide ions can be delineated from the mass spectra, giving insights about changing levels in health and disease. In the examples presented here, the peak intensities of dynorphin B, alpha-neoendorphin and substance P were found to be significantly increased in the dorsolateral, but not the dorsomedial, striatum of animals with severe dyskinesia involving facial, trunk and orolingual muscles (Fig. 5). Furthermore, MALDI IMS revealed a correlation between dyskinesia severity and levels of des-tyrosine alpha-neoendorphin, representing a previously unknown mechanism of functional inactivation of dynorphins in the striatum as the removal of N-terminal tyrosine reduces the dynorphin's opioid-receptor binding capacity(9). This is the first study on neuropeptide characterization in LID using MALDI IMS and the results highlight the potential of the technique for application in all fields of biomedical research.

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    Jove
  • 12.
    Hanrieder, Jörg
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Ljungdahl, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Fälth, Maria
    Eriksson Mammo, Sofie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Andersson, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    L-DOPA-induced dyskinesia is associated with regional increase of striatal dynorphin peptides as elucidated by imaging mass spectrometry2011In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 10, no 10, p. M111.009308-Article in journal (Refereed)
    Abstract [en]

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

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  • 13.
    Hanrieder, Jörg
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
    Nyakas, Adrien
    Naessén, Tord
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
    Proteomic Analysis of Human Follicular Fluid Using an Alternative Bottom-Up Approach2008In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 7, no 1, p. 443-449Article in journal (Refereed)
    Abstract [en]

    Human follicular fluid (hFF) is the in vivo environment of oocytes during follicular maturation in the ovaries. It contains a huge variety of compounds such as, e.g., proteins that might play an important role in follicular development and oocyte growth. Previous proteomic studies on follicular fluid have isolated and already identified a certain number of proteins. Nevertheless, only a small part of proteins present in follicular fluid have been covered so far and a large number have still not been identified. Therefore, the need for new, more resolving, and sensitive approaches in proteome research is evident. We utilized a proteomic setup based on in solution isoelectric focusing (IEF) and reversed-phase nanoliquid chromatography coupled to matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (nano-LC MALDI TOF/TOF MS) for in depth protein analysis of human follicular fluid samples of patients undergoing controlled ovarian hyper stimulation (COH) for in vitro fertilization therapy (IVF). This approach led to the significant identification of 69 proteins, where 32 have not been reported before to be found in human follicular fluid with proteomic methods. Among these findings, at least two relevant compounds essentially involved in hormone secretion regulation during the folliculogenetic process were identified: sex hormone binding globulin (SHBG) and inhibin A (INHA). To confirm these results, both proteins were further validated by immunoassays.

  • 14.
    Hanrieder, Jörg
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Wetterhall, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Enblad, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Neurosurgery. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Hillered, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Neurosurgery. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Temporally resolved differential proteomic analysis of human ventricular CSF for monitoring traumatic brain injury biomarker candidates.2009In: Journal of Neuroscience Methods, ISSN 0165-0270, E-ISSN 1872-678X, Vol. 177, no 2, p. 469-478Article in journal (Refereed)
    Abstract [en]

    A shotgun proteomic approach based on nanoflow liquid chromatography (nanoLC) in conjunction with matrix assisted laser desorption/ionization time of flight tandem mass spectrometry (MALDI TOF MS/MS) was utilized to quantitatively analyze the protein content of consecutive ventricular cerebrospinal fluid (CSF) samples of severe traumatic brain injury (TBI) patients on an individual basis. CSF was acquired from the lateral ventricle 1–9 days after the TBI incident by canula drain to investigate temporally resolved protein changes in three patients that required intracranial pressure monitoring during neurointensive care. The samples were subjected to at once tryptic digestion followed by isobaric tag labeling before multiplexed peptide separation and MS analysis. By using this approach, we were able to follow characteristic changes in protein concentrations over time allowing new conclusions to be drawn about ongoing pathological processes during TBI. Certain suggested protein-biomarker candidates for TBI, like acute phase reactants (APRs), fibrinogens (FIB), cystatin C (CC) or more brain specific proteins like glial fibrillary acid protein (GFAP) and neuron-specific enolase (NSE) were found to be significantly up-regulated which is in strong consistence with previously reported results. This methodology appears to be a promising tool for studying candidate biomarkers of neurovascular and traumatic brain injuries in the neurointensive care setting.

  • 15.
    Hanrieder, Jörg
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Wicher, Grzegorz
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Andersson, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Fex-Svenningsen, Asa
    Institute of Medical Biology, Neurobiology Research, University of Southern Denmark.
    MALDI mass spectrometry based molecular phenotyping of CNS glial cells for prediction in mammalian brain tissue2011In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 401, no 1, p. 135-147Article in journal (Refereed)
    Abstract [en]

    The development of powerful analytical techniques for specific molecular characterization of neural cell types is of central relevance in neuroscience research for elucidating cellular functions in the central nervous system (CNS). This study examines the use of differential protein expression profiling of mammalian neural cells using direct analysis by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). MALDI-MS analysis is rapid, sensitive, robust, and specific for large biomolecules in complex matrices. Here, we describe a newly developed and straightforward methodology for direct characterization of rodent CNS glial cells using MALDI-MS-based intact cell mass spectrometry (ICMS). This molecular phenotyping approach enables monitoring of cell growth stages, (stem) cell differentiation, as well as probing cellular responses towards different stimulations. Glial cells were separated into pure astroglial, microglial, and oligodendroglial cell cultures. The intact cell suspensions were then analyzed directly by MALDI-TOF-MS, resulting in characteristic mass spectra profiles that discriminated glial cell types using principal component analysis. Complementary proteomic experiments revealed the identity of these signature proteins that were predominantly expressed in the different glial cell types, including histone H4 for oligodendrocytes and S100-A10 for astrocytes. MALDI imaging MS was performed, and signature masses were employed as molecular tracers for prediction of oligodendroglial and astroglial localization in brain tissue. The different cell type specific protein distributions in tissue were validated using immunohistochemistry. ICMS of intact neuroglia is a simple and straightforward approach for characterization and discrimination of different cell types with molecular specificity.

  • 16.
    Hanrieder, Jörg
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Zuberovic, Aida
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Surface modified capillary electrophoresis combined with in solution isoelectric focusing and MALDI-TOF/TOF MS: A gel-free multidimensional electrophoresis approach for proteomic profiling — Exemplified on human follicular fluid2009In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1216, no 17, p. 3621-3628Article in journal (Refereed)
    Abstract [en]

    Development of miniaturized analytical tools continues to be of great interest to face the challenges in proteomic analysis of complex biological samples such as human body fluids. In the light of these challenges, special emphasis is put on the speed and simplicity of newly designed technological approaches as well as the need for cost efficiency and low sample consumption. In this study, we present an alternative multidimensional bottom-up approach for proteomic profiling for fast, efficient and sensitive protein analysis in complex biological matrices. The presented setup was based on sample pre-fractionation using microscale in solution isoelectric focusing (IEF) followed by tryptic digestion and subsequent capillary electrophoresis (CE) coupled off-line to matrix assisted laser desorption/ionization time of flight tandem mass spectrometry (MALDI TOF MS/MS). For high performance CE-separation, PolyE-323 modified capillaries were applied to minimize analyte-wall interactions. The potential of the analytical setup was demonstrated on human follicular fluid (hFF) representing a typical complex human body fluid with clinical implication. The obtained results show significant identification of 73 unique proteins (identified at 95% significance level), including mostly acute phase proteins but also protein identities that are well known to be extensively involved in follicular development.

  • 17.
    Hardenborg, Emilia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Taube, Amelie Botling
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Ophthalmology.
    Hanrieder, Jörg
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Andersson, Marit
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Alm, Albert
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Ophthalmology.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Protein content in aqueous humor from patients with pseudoexfoliation (PEX) investigated by capillary-LC MALDI-TOF/TOF MS2009In: PROTEOMICS - Clinical Applications, ISSN 1862-8346, E-ISSN 1862-8354, Vol. 3, no 3, p. 299-306Article in journal (Refereed)
    Abstract [en]

    Analysis of proteins in human body fluids is challenging since the composition of the sample often is rather complex. Here we present a method for analysis of proteins in aqueous humor from two groups of cataract patients, with and without pseudoexfoliation (PEX). Aqueous humor is an extracellular fluid contained in the anterior chamber of the eye between the cornea and iris. The limited volume of sample requires sophisticated analysis techniques. Our method is based on a total tryptic digestion of the sample followed by capillary LC-MALDI MS and MS/MS analysis of the peptides. The method is rapid, efficient and suitable as a complement or alternative to more commonly used methods based on gel electrophoretic experiments. With this method we found and unambiguously identified 30 nonredundant proteins. Proteins found include general transport proteins such as albumin and apolipoprotein A1 but also specific proteins involved in immune response, such as   complement factors. Cystatin C, clusterin, and crystallins were also found. Although the number of proteins was roughly the same in both groups there was a significant difference in their identities. These findings may give some new insights into the pathophysiology of the PEX syndrome.

  • 18.
    Ljungdahl, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Hanrieder, Jörg
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Fälth, Maria
    Unit Cancer Genome Research, Division of Molecular Genetics, German Cancer Research Center (DKFZ), Germany.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Andersson, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Imaging Mass Spectrometry Reveals Elevated Nigral Levels of Dynorphin Neuropeptides in L-DOPA-Induced Dyskinesia in Rat Model of Parkinson's Disease2011In: PLOS ONE, E-ISSN 1932-6203, Vol. 6, no 9, p. e25653-Article in journal (Refereed)
    Abstract [en]

    L-DOPA-induced dyskinesia is a troublesome complication of L-DOPA pharmacotherapy of Parkinson's disease and has been associated with disturbed brain opioid transmission. However, so far the results of clinical and preclinical studies on the effects of opioids agonists and antagonists have been contradictory at best. Prodynorphin mRNA levels correlate well with the severity of dyskinesia in animal models of Parkinson's disease; however the identities of the actual neuroactive opioid effectors in their target basal ganglia output structures have not yet been determined. For the first time MALDI-TOF imaging mass spectrometry (IMS) was used for unbiased assessment and topographical elucidation of prodynorphin-derived peptides in the substantia nigra of a unilateral rat model of Parkinson's disease and L-DOPA induced dyskinesia. Nigral levels of dynorphin B and alpha-neoendorphin strongly correlated with the severity of dyskinesia. Even if dynorphin peptide levels were elevated in both the medial and lateral part of the substantia nigra, MALDI IMS analysis revealed that the most prominent changes were localized to the lateral part of the substantia nigra. MALDI IMS is advantageous compared with traditional molecular methods, such as radioimmunoassay, in that neither the molecular identity analyzed, nor the specific localization needs to be predetermined. Indeed, MALDI IMS revealed that the bioconverted metabolite leu-enkephalin-arg also correlated positively with severity of dyskinesia. Multiplexing DynB and leu-enkephalin-arg ion images revealed small (0.25 by 0.5 mm) nigral subregions with complementing ion intensities, indicating localized peptide release followed by bioconversion. The nigral dynorphins associated with L-DOPA-induced dyskinesia were not those with high affinity to kappa opioid receptors, but consisted of shorter peptides, mainly dynorphin B and alpha-neoendorphin that are known to bind and activate mu and delta opioid receptors. This suggests that mu and/or delta subtype-selective opioid receptor antagonists may be clinically relevant for reducing L-DOPA-induced dyskinesia in Parkinson's disease.

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  • 19.
    Nilsson, Elin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Hanrieder, Jörg
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Larsson, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Proteomic Characterization of IgY Preparations Purified with a Water Dilution Method2008In: Journal of Agricultural and Food Chemistry, ISSN 0021-8561, E-ISSN 1520-5118, Vol. 56, no 24, p. 11638-11642Article in journal (Refereed)
    Abstract [en]

    Antigen-specific chicken IgY antibodies have been used for oral immunotherapy as an alternative orcomplement to antibiotics in several studies. The water dilution (WD) method has several advantagesfor purifying IgY. It is rapid, efficient, suitable for large-scale production, and nothing but water isadded. The water-soluble fraction contains other proteins and lipids besides IgY. The protein contentwas characterized by two-dimensional gel electrophoresis (2DGE) and nanoflow liquid chromatographycoupled offline to matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry(nanoLC-MALDI TOF/TOF MS). Protein analysis was complicated due to the large dynamicconcentration range, but 26 proteins could be identified. The relative protein concentrations in differentbatches were very similar according to protein patterns on 1D gels and protein concentrationdeterminations. Thus, the purification method has a high reproducibility. The concentrations ofcholesterols and triglycerides were low and should not have an effect on the plasma levels of treatedpatients. Purification of IgY for oral use with WD is therefore a recommended method.

  • 20. Ramström, Margareta
    et al.
    Zuberovic, Aida
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Grönwall, Caroline
    Department of Molecular Biotechnology, School of Biotechnology, Royal Institute of Technology (KTH), Stockholm, Sweden.
    Hanrieder, Jörg
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Hober, Sophia
    Department of Proteomics, School of Biotechnology, AlbaNova University Center, Royal Institute of Technology (KTH), Stockholm, Sweden,.
    Development of affinity columns for the removal of high-abundance proteins in cerebrospinal fluid2009In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 52, no Pt 2, p. 159-166Article in journal (Refereed)
    Abstract [en]

    Various approaches for removal of high-abundance components in body fluids are currently available. While most methods are constructed for plasma depletion, there is a need for body-fluid-specific strategies. The aim of the present study was to design an affinity matrix suitable for the depletion of high-abundance proteins in CSF (cerebrospinal fluid). Hence, molecules with specific affinity towards proteins present at high concentration in CSF were desired. Affibody molecules are specific binders of small size that have shown high stability under various conditions and are therefore good candidates for such a matrix. The protein composition in CSF resembles that in plasma. However, 20% of the proteins are brain-derived and are therefore present in higher proportions in CSF than in plasma, whereas larger plasma-derived proteins are less abundant in CSF. Therefore five high-abundance CSF proteins were chosen for the design of a CSF-specific depletion setup. Affibody molecules with specificity towards HSA (human serum albumin), IgG, transferrin and transthyretin were combined in an affinity column. In addition, polyclonal antibodies against cystatin C were coupled to chromatographic beads and packed in a separate column. Highly reproducible and efficient removal of the five target proteins was observed. The proportion of depleted proteins were estimated to be 99, 95, 74, 92 and 83% for HSA, IgG, transferrin, transthyretin and cystatin C respectively. SDS/PAGE analysis was used for monitoring and identifying proteins in native CSF, depleted CSF samples and the captured fractions. Moreover, shotgun proteomics was used for protein identification in native as well as depleted CSF and the achieved data were compared. Enhanced identification of lower-abundance components was observed in the depleted fraction, in terms of more detected peptides per protein.

  • 21.
    Taube, Amelie Botling
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Ophthalmology. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Hardenborg, Emilia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Wetterhall, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Artemenko, Konstantin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Hanrieder, Jörg
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Andersson, Marit
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Alm, Albert
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Ophthalmology.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Proteins in aqueous humor from cataract patients with and without pseudoexfoliation syndrome2012In: European journal of mass spectrometry, ISSN 1469-0667, E-ISSN 1751-6838, Vol. 18, no 6, p. 531-541Article in journal (Refereed)
    Abstract [en]

    The aim of this study was to investigate the protein content in aqueous humor in eyes with and without pseudoexfoliations (PEX) and to evaluate the quantitative proteomics method, isobaric tagging for relative and absolute protein quantification (iTRAQ), in combination with two separation methods followed by matrix-assisted Laser desorption/ionization (MALDI) mass spectrometry and tandem mass spectrometry (MS/MS). During cataract surgery, samples of aqueous humor were collected from 20 eyes with PEX and from 18 control eyes. The relative concentrations of proteins in the pooled samples of ten PEX eyes and eight controls were evaluated after trypsin digestion and labeling of the peptides with (iTRAQ) reagent. Two separation methods, Liquid chromatography (LC) and capillary electrophoresis (CE) were used, followed by MALDI mass spectrometry and MS/MS. Furthermore, 1D gel electrophoresis was performed on the remaining ten pooled PEX samples and ten control samples. The gel material was separated by nano-liquid chromatography (nano-LC) followed by Linear-ion-trap quadrupole Fourier transformation ion cyclotron resonance (FT-ICR). Fifty four proteins were identified in the LC runs and 24 with CE. The relative concentrations of beta-crystallines B2 and S were raised and those of angiotensinogen and osteopontin lowered in the PEX sample compared to the control. The trends regarding beta-crystallines B2, angiotensinogen and osteopontin were confirmed by the 1D gel electrophoresis.

  • 22.
    Wetterhall, Magnus
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
    Zuberovic, Aida
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
    Hanrieder, Jörg
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
    Assessment of the partitioning capacity of high abundant proteins in human cerebrospinal fluid using affinity and immunoaffinity subtraction spin columns.2010In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 878, no 19, p. 1519-1530Article in journal (Refereed)
    Abstract [en]

    The performance of three different affinity and immunoaffinity subtraction spin columns was investigated for the removal of the most abundant proteins in human cerebrospinal fluid (CSF). A pool of human CSF was processed with the spin columns and both the bound and flow through fractions were compared with each other and with intact CSF using 1D gel electrophoresis and nanoLC-MALDI-TOF/TOF-MS analysis. MASCOT MS/MS ionscores were compared before and after processing with the columns. The non-specific co-removal of proteins bound to the high abundant proteins, so called "sponge effect" was also examined for each spin column. The reproducibility of one of the spin columns, ProteomeLab IgY-12 proteome partitioning spin column, was further investigated by isobaric tags for relative and absolute quantification (iTRAQ) labeling and MS/MS analysis. Overall, 173 unique proteins were identified on a 95% MudPIT confidence scoring level. For all three spin columns, the number of proteins identified and their MASCOT scores were increased up to 10 times. The largest degree of non-specific protein removal was observed for a purely affinity based albumin removal column, where 28 other proteins also were present. The ProteomeLab IgY-12 proteome partitioning spin column showed very high reproducibility when combined with iTRAQ labeling and MS/MS analysis. The combined relative standard deviation (R.S.D.) for the high abundant protein removal, iTRAQ labeling and nanoLC-MALDI-TOF/TOF-MS analysis was less than 17.5%.

  • 23.
    Zuberovic, Aida
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Hanrieder, Jörg
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Wetterhall, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    CE-MALDI-MS/MS for Proteomic Profiling and Multiplexed Quantification of Human Cerebrospinal Fluid (CSF)2007Conference paper (Other academic)
  • 24.
    Zuberovic, Aida
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
    Hanrieder, Jörg
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
    Wetterhall, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
    Proteome profiling of human cerebrospinal fluid: exploring the potential of capillary electrophoresis with surface modified capillaries for analysis of complex biological samples.2008In: European journal of mass spectrometry, ISSN 1469-0667, E-ISSN 1751-6838, Vol. 14, no 4, p. 249-260Article in journal (Refereed)
    Abstract [en]

    A bottom-up proteomic approach, based on capillary electrophoresis (CE) in combination with matrix- assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-ToF/ToF MS), was used to analyze immunoaffinity depleted human cerebrospinal fluid (CSF) and compare it with a non-depleted sample. After enzymatic digestion and desalting, the tryptic peptides were separated by CE using PolyE-323 modified capillaries and fractionated off-line onto MALDI target plates for further analysis by MALDI-MS and MS/MS. The protein profile of the depleted sample was compared with non depleted CSF. Overall, 85 proteins were identified with 95% significance in both samples. The significance scores for proposed biomarkers, such as amyloid-like protein 1 precursor, could be increased up to 12 times after the depletion. Other proteins, often suggested to be related to neurodegenerative diseases, like amyloid beta A4 protein precursor, superoxide dismutase and apolipoprotein E precursor could only be found in the depleted CSF samples. The effect of a derivatization of tryptic peptides with 2- methoxy-4,5-dihydro-1H-imidazole reagent for protein identification with MS was also employed to increase the number of identified proteins and the sequence coverages. The results presented in this study illustrate the benefit of combining a sample pre-fractionation step and a label's ability to enhance the ionization efficiency with the potential of CE using PolyE-323 modified capillaries in the analysis of complex samples. The straight-forward approach that provides speed and simplicity resulting in high-resolution separations and low sample consumption represents an easily applicable separation technique that can serve as a complement to other currently existing analytical approaches needed in modern proteomic analysis of clinically relevant samples.

  • 25.
    Zuberovic, Aida
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Wetterhall, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Hanrieder, Jörg
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    CE MALDI-TOF/TOF MS for multiplexed quantification of proteins in human ventricular cerebrospinal fluid2009In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 30, no 10, p. 1836-1843Article in journal (Refereed)
    Abstract [en]

    CE, interfaced off-line to MALDI-TOF/TOF MS, was for the first time used to quantitatively monitor the protein content in complex   biological and clinical samples with iTRAQ (TM) labeling. The   usefulness and advantage of iTRAQ (TM) labeling, in combination, with   CE MALDI-TOF/TOF MS is demonstrated on mixtures of protein standards and by a case study on human ventricular cerebrospinal fluid samples collected from a patient with traumatic brain injury during patient recovery. Mixtures of five standard proteins were initially analyzed to optimize the experimental conditions for the CE MALDI-MS and MS/MS  analysis. The interactions of proteins and peptides with the capillary   inner wall during CE separation were minimized using PolyE-323 modified  capillaries. The analysis of the ventricular cerebrospinal fluid   samples yielded 43 significantly (p < 0.05 MudPIT scoring) identified   proteins that could be quantitatively monitored over time. The identified changes in protein levels for several of these proteins are well in line with the reports from previous studies on protein patterns that could be related to the post-traumatic processes of traumatic brain injury. This study shows that the presented approach, combining  isobaric tags with CE MALDI-TOF/TOF MS, is a useful choice for quantitative proteomic analysis.

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