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  • 1.
    Amirkhani, Ardeshir
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Wetterhall, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Nilsson, Stefan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Danielsson, Rolf
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Comparison between different sheathless electrospray emitter configurations regarding the performance of nanoscale liquid chromatography time-of-flight mass spectrometry analysis2004In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1033, no 2, p. 257-266Article in journal (Refereed)
    Abstract [en]

    Four different sheathless electrospray ionization (ESI) configurations were investigated for a nano liquid chromatography (LC) system. The studied configurations were: a column with an integrated emitter, with the ESI potential applied before or after the column, and a column with separate emitter, with the ESI voltage applied at a union before the emitter or at the emitter tip. The results indicates that the efficiency of the LC system is rather independent of the configuration when using 95 μm i.d. columns, acetic mobile phase and standard peptides as a sample. Introduction of post column dead volume seems not to be a critical issue at least with flow rates down to 600 nl/min.

  • 2.
    Bergquist, Jonas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Palmblad, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science.
    Wetterhall, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Håkansson, Per
    Markides, Karin E
    Peptide Mapping of Proteins in Human Body Fluids using Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry2002In: Mass spectrometry reviews (Print), ISSN 0277-7037, E-ISSN 1098-2787, Vol. 21, no 1, p. 2-15Article in journal (Refereed)
    Abstract [en]

    Human body fluids have been rediscovered in the postgenomic era as great sources of biological markers and perhaps particularly as sources of potential protein biomarkers of disease. Analytical tools that allow rapid screening, low sample consumption, and accurate protein identification are of great importance in studies of complex biological samples and clinical diagnosis. Mass spectrometry is today one of the most important analytical tools with applications in a wide variety of fields. One of the fastest growing applications is in proteomics, or the study of protein expression in an organism. Mass spectrometry has been used to find post-translational modifications and to identify key functions of proteins in the human body. In this study, we review the use of human body fluids as sources for clinical markers and present new data that show the ability of Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) to identify, and characterize proteins in four human body fluids: plasma, cerebrospinal fluid (CSF), saliva, and urine. The body fluids were tryptically digested without any prior separation, purification, or selection, and the digest was introduced into a 9.4 T FTICR mass spectrometer by direct-infusion electrospray ionization (ESI). Even though these samples represent complex biological mixtures, the described method provides information that is comparable with traditional 2D-PAGE data. The sample consumption is extremely low, a few microliters, and the analysis time is only a few minutes. It is, however evident that the separation of proteins and/or peptides must be included in the methodology in order to detect low-abundance proteins and other proteins of biological relevance.

  • 3.
    Dahlin, Andreas P
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Microsystems Technology.
    Hjort, Klas
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Microsystems Technology.
    Hillered, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Neurosurgery.
    Sjödin, Marcus O D
    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.
    Wetterhall, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Quantification of Proteins Adsorbed to Surface Modified and Non-Modified Microdialysis Membranes using on-Surface Enzymatic Digestion (oSED) iTRAQ-MALDI-TOF/TOF MS2012In: 60th ASMS Conference on Mass Spectrometry and Allied Topics, May 20 - 24, Vancouver, Canada, 2012Conference paper (Refereed)
  • 4.
    Dahlin, Andreas P
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Microsystems Technology.
    Hjort, Klas
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Microsystems Technology.
    Hillered, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Neurosurgery.
    Sjödin, Marcus O.D.
    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.
    Wetterhall, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Multiplexed quantification of proteins adsorbed to surface-modified and non-modified microdialysis membranes2012In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 402, no 6, p. 2057-2067Article in journal (Refereed)
    Abstract [en]

    A simple and straightforward method for discovery and quantification of proteins adsorbed onto delicate and sensitive membrane surfaces is presented. The adsorbed proteins were enzymatically cleaved while still adsorbed onto the membranes using an on-surface enzymatic digestion (oSED). This was followed by isobaric tagging, nanoliquid chromatography, and tandem mass spectrometry. Protein adsorption on tri-block copolymer Poloxamer 407 surface-modified microdialysis (MD) membranes were compared with protein adsorption on unmodified MD membranes. Ventricular cerebrospinal fluid (vCSF) kept at 37 °C was used as sample matrix. In total, 19 proteins were quantified in two biological replicates. The surface-modified membranes adsorbed 33% less proteins than control membranes and the most abundant proteins were subunits of hemoglobin and clusterin. The adsorption of clusterin on the modified membranes was on average 36% compared to control membranes. The most common protein in vCSF, Albumin, was not identified adsorbed to the surface at all. It was also experimentally verified that oSED, in conjunction with tandem mass spectrometry can be used to quantify femtomole amounts of proteins adsorbed on limited and delicate surfaces, such as MD membranes. The method has great potential and can be used to study much more complex protein adsorption systems than previously reported.

  • 5.
    Dahlin, Andreas P.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Microsystems Technology.
    Wetterhall, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
    Caldwell, Karin D.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
    Larsson, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
    Hillered, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Neurosurgery.
    Hjort, Klas
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Microsystems Technology.
    Methodological aspects on microdialysis protein sampling and quantification in biological fluids: an in vitro study on human ventricular CSF.2010In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 82, no 11, p. 4376-4385Article in journal (Refereed)
    Abstract [en]

    There is growing interest in sampling of protein biomarkers from the interstitial compartment of the brain and other organs using high molecular cutoff membrane microdialysis (MD) catheters. However, recent data suggest that protein sampling across such MD membranes is a highly complex process that needs to be further studied. Here, we report three major improvements for microdialysis sampling of proteins in complex biological matrixes. The improvements in this in vitro study using human ventricular cerebrospinal fluid as the sample matrix include increased fluid recovery control, decreased protein adsorption on the microdialysis membrane and materials, and novel quantitative mass spectrometry analysis. Dextrans in different concentrations and sizes were added to the perfusion fluid. It was found that dextrans with molecular mass 250 and 500 kDa provided a fluid recovery close to 100%. An improved fluid recovery precision could be obtained by self-assembly triblock polymer surface modification of the MD catheters. The modified catheters also delivered a significantly increased extraction efficiency for some of the investigated proteins. The final improvement was to analyze the dialysates with isobaric tagged (iTRAQ) proteomics, followed by tandem mass spectrometric analysis. By using this technique, 48 proteins could be quantified and analyzed with respect to their extraction efficiencies. The novel aspects of microdialysis protein sampling, detection, and quantification in biological fluids presented in this study should be considered as a first step toward better understanding and handling of the challenges associated with microdialysis sampling of proteins. The next step is to optimize the developed methodology in vivo.

  • 6.
    Elhamili, Anisa
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Samuelsson, Jörgen
    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.
    Optimizing the extraction, separation and quantification of tricyclic antidepressant drugs in human plasma with CE-ESI-TOF-MS using cationic coated capillaries2011In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 32, no 6-7, p. 647-658Article in journal (Refereed)
    Abstract [en]

    In this study, the extraction and CE-ESI-TOF-MS analysis of tricyclic antidepressant (TCA) drugs imipramine, desipramine, clomipramine and norclomipramine in human plasma has been optimized. The CE capillaries were modified with ω-iodo-alkyl ammonium salt (M7C4I coating) to reduce analyte adsorption to the silica wall. The use of a strong cation exchange (SCX) solid-phase extraction (SPE) column specifically designed for the extraction of basic drug species from biofluids gave very clean extracts with high and reproducible recoveries. The extraction recoveries were ranging between 87 and 91% with % RSD values of 0.5-1.7% (n=3). The obtained strong cation exchange-SPE extracts of the TCA in human plasma only contained the analytes of interest. The optimized CE separation conditions were obtained by adding ACN and acetic acid to the sample while using an aqueous BGE. The CE-ESI-TOF-MS analysis was performed within 6min for all TCA analytes under the optimized condition with peak efficiencies up to 1.4×105plates/m and an average % RSD of the migration times of the analytes of 0.3% (n=5). The presented method can readily be used for the extraction and quantification of basic drug species in human biological fluids and in pharmaceutical formulations.

  • 7.
    Elhamili, Anisa
    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.
    Arvidsson, Björn
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Sebastiano, Roberto
    Righetti, Pier Giorgio
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Rapid capillary electrophoresis time-of-flight mass spectrometry separations of peptides and proteins using a monoquaternarized piperazine compound (M7C4l) for capillary coatings2008In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 29, no 8, p. 1619-1625Article in journal (Refereed)
    Abstract [en]

    A monoquaternarized piperazine, 1-(4-iodobutyl) 4-aza-1-azoniabicyclo[2,2,2] octane iodide (M7C4I), has been evaluated as a surface derivatization reagent for CE in combination with TOF MS for the analysis of proteins, peptides, and protein digests. The M7C4I piperazine, at alkaline pH, forms a covalent bond via alkylation of the ionized silanols producing a cationic surface with a highly stable and reversed EOF. The obtained surface yields rapid separations (less than 5 min) of peptides and proteins at acidic pH with high separation efficiencies (up to 1.1 X 10(6) plates/m for peptides and up to 1.8 x 10(6) plates/m for proteins) and no observed bleeding of the coating reagent into the mass spectrometer. The simplicity of the coating procedure also enables fast (2 min) regeneration of the surface, if necessary. This is useful in the analysis of complex samples in order to prevent possible memory effects. The potential of using M7C4I-coated capillaries for MS analysis of complex samples is demonstrated by the separation of peptides, proteins, and protein digests. Even more, the spectacular thing in which large intact proteins with molecular masses over 0.5 MDa could be separated. The coating showed good ability to handle these large proteins with high efficiency and retained peak shape as demonstrated by separation of IgG(1) (150 kDa) and thyroglobulin (669 kDa).

  • 8.
    Elhamili, Anisa
    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.
    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.
    Puerta, Angel
    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.
    Westerlund, Douglas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical 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.
    The effect of sample salt additives on capillary electrophoresis analysis of intact proteins using surface modified capillaries.2009In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1216, no 17, p. 3613-3620Article in journal (Refereed)
    Abstract [en]

    The effect of adding alkali salts to protein samples for capillary electrophoretic (CE) analysis of intact proteins was studied. A high degree of peak stacking, even for large proteins, was found to occur when alkali salts were added to the sample. The addition of salt to the protein sample promotes a strong improvement in the peak efficiency of individual proteins giving up to 2.1 x 10(6) apparent plates/m. The concentration of salt required in the sample to reach optimal peak efficiency show dependency on both the molecular weight and molar concentration of the protein. However, adding salt will, at a sufficiently high concentration, cause a mixture of proteins to co-migrate to one very sharp peak. The observed sample stacking effect was obtained with a number of different surface modified silica capillaries indicating a general phenomenon and not surface coating specific.

  • 9.
    Elhamili, Anisa
    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.
    Sjödin, Marcus O.D.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Sebastiano, Roberto
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Analysis of peptides using N-methylpolyvinylpyridium as silica surface modifier for CE-ESI-MS2010In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 31, no 7, p. 1151-1156Article in journal (Refereed)
    Abstract [en]

    In this study, the N-methylpolyvinylpyridinuim polymer has for the first time been used as a silica surface modifier for CE in combination with ESI MS (CE-ESI-MS). The compatibility for ESI-MS was demonstrated by the analysis of peptides and protein digests. The N-methylpolyvinylpyridium surface interacts electrostatically with the ionized silanol groups, giving a cationic surface with a reversed EOF. The surface modifier gave rapid and repeatable separations of peptides, proteins and protein digests at acidic pH for more than 4 h of continuous use. The CE separation yielded peak efficiencies of up to 4.3 x 10(5) plates/m. The surface coating is highly compatible with ESI and facilitates the separation and analysis of complex peptide mixtures as shown by the analysis of BSA digests.

  • 10.
    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.

  • 11.
    Johannesson, Nina
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
    Olsson, Louise
    Bäckström, Daniel
    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.
    Danielsson, Rolf
    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.
    Screening for biomarkers in plasma from patients with gangrenous phlegmonous appendicitis using CE and CEC in combination with MS2007In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 28, no 9, p. 1435-1443Article in journal (Refereed)
    Abstract [en]

    Today a high degree of "false" appendicitis diagnoses are occurring. In this study, a screening experiment of biomarkers of two different kinds of appendicitis, gangrenous and phlegmonous, were conducted with CE and CEC coupled to MS. Plasma samples were obtained from patients pre- and post-surgery. A large amount of data was generated to be able to compare them, and chemometrics tools were utilized to visualize the differences. Indicative patterns were found for both pre- and post-surgery of the two types of inflammation as well as between them. The divergences were traced back to the MS peaks obtained in the CE- and CEC-MS setups as possible biomarkers for the two forms of appendicitis.

  • 12.
    Johannesson, Nina
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Wetterhall, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Markides, Karin E.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Monomer surface modifications for rapid peptide analysis by capillary electrphoresis and capillary electrochromatography coupled to electrospray ionization mass spectrometry2004In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 25, no 6, p. 809-816Article in journal (Refereed)
    Abstract [en]

    In this study, positively charged alkylaminosilyl monomers were used to modify the inner surface of fused silica capillaries, which subsequently were employed in capillary electrophoresis (CE) and capillary electrochromatography (CEC). The obtained surfaces yield a reversed electroosmotic flow (EOF) and have varying carbon chain lengths, that interact with the analytes and give chromatographic retention. The coating procedure is very simple and fast. The performance of the modified capillaries was evaluated regarding pH influence on EOF and chromatographic interactions. The experiments were conducted with UV and mass spectrometry (MS) and applied to the separation of various neuropeptides. The derivatized surfaces showed a linear (R2 ∼0.99) pH dependence with isoelectric points (pI) at 8.6–8.8. Rapid separations of peptide standards and a protein digest with efficiencies as high as 5×105 plates/m were performed.

  • 13.
    Loov, Camilla
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Neurosurgery.
    Shevchenko, Ganna
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Nadadhur, Aishwarya Geeyarpuram
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Neurosurgery.
    Clausen, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Neurosurgery.
    Hillered, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Neurosurgery.
    Wetterhall, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Erlandsson, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Neurosurgery.
    Identification of Injury Specific Proteins in a Cell Culture Model of Traumatic Brain Injury2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 2, p. e55983-Article in journal (Refereed)
    Abstract [en]

    The complicated secondary molecular and cellular mechanisms following traumatic brain injury (TBI) are still not fully understood. In the present study, we have used mass spectrometry to identify injury specific proteins in an in vitro model of TBI. A standardized injury was induced by scalpel cuts through a mixed cell culture of astrocytes, oligodendrocytes and neurons. Twenty-four hours after the injury, cell culture medium and whole-cell fractions were collected for analysis. We found 53 medium proteins and 46 cell fraction proteins that were specifically expressed after injury and the known function of these proteins was elucidated by an extensive literature survey. By using time-lapse microscopy and immunostainings we could link a large proportion of the proteins to specific cellular processes that occur in response to trauma; including cell death, proliferation, lamellipodia formation, axonal regeneration, actin remodeling, migration and inflammation. A high percentage of the proteins uniquely expressed in the medium after injury were actin-related proteins, which normally are situated intracellularly. We show that two of these, ezrin and moesin, are expressed by astrocytes both in the cell culture model and in mouse brain subjected to experimental TBI. Interestingly, we found many inflammation-related proteins, despite the fact that cells were present in the culture. This study contributes with important knowledge about the cellular responses after trauma and identifies several potential cell-specific biomarkers.

  • 14.
    Lööv, Camilla
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Neurosurgery.
    Shevchenko, Ganna
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Hillered, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Neurosurgery.
    Wetterhall, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Erlandsson, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Neurosurgery.
    Identification of unique proteins after injury in a cell culture model of TBI2012In: Brain Injury, ISSN 0269-9052, E-ISSN 1362-301X, Vol. 26, no 4-5, p. 487-488Article in journal (Other academic)
  • 15.
    Nilsson, Stefan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Wetterhall, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Nyholm, Leif
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Markides, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    A simple and robust conductive graphite coating for sheathless electrospray emitters used in capillary electrophoresis/mass spectrometry2001In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 15, no 21, p. 1997-2000Article in journal (Refereed)
    Abstract [en]

    A graphite-polyimide mixture was used as a conductive coating for sheathless electrospray emitters. The coating procedure described is simple and inexpensive compared to previously described methods. An investigation of the stability of the conductive coating carried out by electrochemical methods revealed good performances during oxidative stress. In addition, no decrease in emitter performance was seen during continuous electrospray in the positive electrospray mode for two weeks. Fast capillary electrophoresis with attomole sensitivity demonstrated the excellent performance of the described sheathless interface when used in conjunction with an orthogonal time-of-flight mass spectrometer. The overall simplicity, stability and low cost of this type of sheathless emitter make the described approach highly suitable for any on-column coupling of low flow rate separation techniques to a mass spectrometer.

  • 16.
    Palmblad, Magnus
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science. Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Wetterhall, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Markides, Karin E
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Håkansson, Per
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Analysis of Enzymatically Digested Proteins and Protein Mixtures using a 9.4 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometer2000In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 14, no 12, p. 1029-1034Article in journal (Refereed)
    Abstract [en]

    A commercially available 9.4 Tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer was applied in the analysis of tryptic digests of protein mixtures without any separation. First, the method was demonstrated on a mixture of tryptic digests of equine cytochrome c, equine myoglobin and bovine serum albumin. The same method was then applied to human plasma from a healthy blood donor. Computer programs were employed to simplify analysis of the complex spectra. The 2745 peaks in the human plasma electrospray ionization FTICR spectrum could be reduced to 1165 isotopic clusters and 669 unique masses. Out of these, 82 masses matched tryptic fragments of serum albumin with mass measurement errors less than 10 ppm, covering 93% of the sequence. Another 16 masses were assigned to tryptic fragments of transferrin, covering 41% of the sequence on the 10 ppm mass measurement error level (14 within 2 ppm). The mass measurement errors were approximately normal distributed with a standard deviation of 1.7 ppm. This demonstrates the feasibility of combining the ultra-high mass resolving power and accuracy of FTICR mass spectrometry with automated computer analysis for investigating complex biological matrices.

  • 17.
    Pettersson Dahlin, Andreas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Wetterhall, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Liljegren, Gustav
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Bergström, Sara K.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Andrén, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Nyholm, Leif
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Materials Chemistry, Materials Chemistry.
    Markides, Karin E
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Capillary electrophoresis coupled to mass spectrometry from a polymer modified poly(dimethylsiloxane) microchip with an integrated graphite electrospray tip2005In: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 130, no 2, p. 193-199Article in journal (Refereed)
    Abstract [en]

    Hybrid capillary-poly(dimethysiloxane) (PDMS) microchips with integrated electrospray ionization (ESI) tips were directly fabricated by casting PDMS in a mould. The shapes of the emitter tips were drilled into the mould, which produced highly reproducible three-dimensional tips. Due to the fabrication method of the microfluidic devices, no sealing was necessary and it was possible to produce a perfect channel modified by PolyE-323, an aliphatic polyamine coating agent. A variety of different coating procedures were also evaluated for the outside of the emitter tip. Dusting graphite on a thin unpolymerised PDMS layer followed by polymerisation was proven to be the most suitable procedure. The emitter tips showed excellent electrochemical properties and durabilities. The coating of the emitter was eventually passivated, but not lost, and could be regenerated by electrochemical means. The excellent electrochemical stability was further confirmed in long term electrospray experiments, in which the emitter sprayed continuously for more than 180 h. The PolyE-323 was found suitable for systems that integrate rigid fused silica and soft PDMS technology, since it simply could be applied successfully to both materials. The spray stability was confirmed from the recording of a total ion chromatogram in which the electrospray current exhibited a relative standard deviation of 3.9% for a 30 min run. CE-ESI-MS separations of peptides were carried out within 2 min using the hybrid PDMS chip resulting in similar efficiencies as for fused silica capillaries of the same length and thus with no measurable band broadening effects, originating from the PDMS emitter.

  • 18.
    Samskog, Jenny
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Bergström, Sara K.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Jönsson, Mats
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Solid State Electronics.
    Klett, Oliver
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Wetterhall, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Markides, Karin E
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    On-column polymer-imbedded graphite inlet electrode for capillary electrophoresis coupled on-line with flow injection analysis in a poly(dimethylsiloxane) interface2003In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 24, no 11, p. 1723-1729Article in journal (Refereed)
    Abstract [en]

    A method for coupling an electrophoretic driven separation to a liquid flow, using conventional fused-silica capillaries and a soft polymeric interface is presented. A novel design of the electrode providing high voltage to the electrophoretic separation was also developed. The electrode consisted of a conductive polyimide/graphite imbedded coating immobilized onto the capillary electrophoresis (CE) column inlet. This integrated electrode gave the same separation performance as a commonly used platinum electrode. The on-column electrode also showed good electrochemical stability in chronoamperometric experiments. In addition, with this electrode design, the electrode position relative to the inlet end of the CE column will always be constant and well defined. The on-line flow injection analysis (FIA)-CE system was used with electrospray ionization (ESI)-time of flight (TOF)-mass spectrometry detection. The preparation of the PDMS (poly(dimethylsiloxane)) interface for FIA-CE is described in detail and used for initial tests of the on-column polymer-imbedded graphite inlet electrode. In this interface, a pressure-driven liquid flow, a make up CE electrolyte and a CE column inlet meet in a two-level cross (95 μm ID) in the PDMS structure, enabling independent flow characterization.

  • 19.
    Samskog, Jenny
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Wetterhall, Magnus
    Jacobsson, Sven P
    Markides, Karin E
    Optimization of capillary electrophoresis conditions for coupling to a mass spectrometer via a sheathless interface2000In: J Mass Spectrom, Vol. 35, no 7, p. 919-924Article in journal (Refereed)
  • 20.
    Shepherd, Tyson R
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. MIT, Dept Biol Engn, Cambridge, MA 02139 USA..
    Du, Liping
    Vanderbilt Univ, Med Ctr, Dept Pharmacol, Nashville, TN 37232 USA.;Vanderbilt Univ, Med Ctr, Ctr Quantitat Sci, Nashville, TN 37232 USA..
    Liljeruhm, Josefine
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Samudyata,
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Wang, Jinfan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Stanford Univ, Sch Med, Dept Biol Struct, Stanford, CA 94305 USA..
    Sjödin, Marcus O.D.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Swedish Toxicol Sci Res Ctr Swetox, S-15136 Sodertalje, Sweden..
    Wetterhall, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. GE Healthcare Biosci, Life Sci, S-75184 Uppsala, Sweden..
    Yomo, Tetsuya
    East China Normal Univ, Inst Biol & Informat Sci, Sch Comp Sci & Software Engn, Sch Life Sci, Shanghai 200062, Peoples R China..
    Forster, Anthony C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Vanderbilt Univ, Med Ctr, Dept Pharmacol, Nashville, TN 37232 USA..
    De novo design and synthesis of a 30-cistron translation-factor module2017In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, no 18, p. 10895-10905Article in journal (Refereed)
    Abstract [en]

    Two of the many goals of synthetic biology are synthesizing large biochemical systems and simplifying their assembly. While several genes have been assembled together by modular idempotent cloning, it is unclear if such simplified strategies scale to very large constructs for expression and purification of whole pathways. Here we synthesize from oligodeoxyribonucleotides a completely de-novo-designed, 58-kb multigene DNA. This BioBrick plasmid insert encodes 30 of the 31 translation factors of the PURE translation system, each His-tagged and in separate transcription cistrons. Dividing the insert between three high-copy expression plasmids enables the bulk purification of the aminoacyl-tRNA synthetases and translation factors necessary for affordable, scalable reconstitution of an in vitro transcription and translation system, PURE 3.0.

  • 21.
    Shevchenko, Ganna
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Musunuri, Sravani
    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.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Comparison of Extraction Methods for the Comprehensive Analysis of Mouse Brain Proteome using Shotgun-based Mass Spectrometry2012In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 11, no 4, p. 2441-2451Article in journal (Refereed)
    Abstract [en]

    This study compares 16 different extraction methods for the comprehensive extraction of mouse brain proteome in combination with "shotgun"-based mass spectrometry (MS). Membrane proteins (MPs) are responsible for a large part of the regulatory functions of the cell and are therefore of great interest to extract and analyze. Sixteen protein extraction protocols were evaluated in regards to protein yield and number of identified proteins with emphasis on MPs. The extracted proteins were delipidated, on-filter digested, and analyzed by reversed phase nanoliquid chromatography (RP-nanoLC) in combination with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using a 7 T hybrid LTQ: FT mass spectrometer. Detergent-based lysis buffers showed higher efficiencies and yields in the extraction of proteins from the brain tissue compared to solubilization with organic solvents or organic acids. The detergent octyl-beta-D-glucopyranoside gave the highest number of identified proteins (541) as well as numbers and percentages of identified MPs (29%). Detergent-based protocols are the best sample preparation tools for central nervous system (CNS) tissue and can readily be applied to screen for candidate biomarkers of neurological diseases.

  • 22.
    Shevchenko, Ganna
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Sjödin, Marcus O.D.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Malmström, David
    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.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Cloud-point extraction and delipidation of porcine brain proteins in combination with bottom-up mass spectrometry approaches for proteome analysis2010In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 9, no 8, p. 3903-3911Article in journal (Refereed)
    Abstract [en]

    In this study, temperature-induced phase fractionation also known as cloud-point extraction (CPE) with the nonionic surfactant Triton X-114 was used to simultaneously extract hydrophobic and hydrophilic proteins from porcine brain tissue. Various protein precipitation/delipidation procedures were investigated to efficiently remove lipids and detergents while retaining maximum protein recoveries. The best performing delipidation method was then used in combination with CPE to compare three different mass spectrometry (MS) based "bottom-up" proteomic approaches for protein analysis of the porcine brain. In the first approach, the intact proteins were initially separated by one-dimensional (1D) gel electrophoresis. The excised protein bands were digested with trypsin, and the peptides were separated by reversed phase nanoliquid chromatography (RP-nanoLC) followed by electrospray ionization (ESI) tandem mass spectrometry (MS/MS) analysis. The other bottom-up proteomic approaches were based on first enzymatical digestion of the proteins followed by RP-nanoLC separation in combination with matrix assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS) or on the combination of in-solution isoelectric focusing (IEF) with ESI-nanoLC-MS/MS of the IEF separated peptides. In total, we found and unambiguously identified 331 unique proteins. The overlap between different techniques was about 10%, showing that the use of multiple proteomic approaches is beneficial to yield a better coverage of the proteome. Furthermore, the overlap between the CPE extracted hydrophilic and hydrophobic proteins was rather small (9-16%), indicating an efficient sample preparation technique to extract and separate hydrophilic and hydrophobic proteins from brain tissue. The percentage of identified membrane proteins was 27%, which is in accordance to the fact that about one-third of all genes in various organisms encode for this class of proteins. The results indicate that cloud point extraction is a promising sample preparation tool, which allows simultaneous in depth studies of brain derived membrane proteins as well as hydrophilic proteins. This technique can be very useful when studying human central nervous system (CNS) tissue or animal models of neurological diseases.

  • 23.
    Shevchenko, Ganna
    et al.
    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.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Höglund, Kina
    Andersson, Lars I
    Kultima, Kim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Longitudinal characterization of the brain proteomes for the tg2576 amyloid mouse model using shotgun based mass spectrometry2012In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 11, no 12, p. 6159-74Article in journal (Refereed)
    Abstract [en]

    Neurodegenerative disorders are often defined pathologically by the presence of protein aggregates, such as amyloid plaques composed of β-amyloid (Aβ) peptide in Alzheimer's disease. Such aggregates are the result of abnormal protein accumulation and may lead to neuronal dysfunction and cell death. In this study, APPSWE transgenic mice (Tg2576), which overexpress the Swedish mutated form of human amyloid precursor protein (APP), were used to study the brain proteome associated with amyloid plaque deposition. The major aim of the study was to map and compare the Tg2576 model brain proteome profiles during pathology progression using a shotgun approach based on label free quantification with mass spectrometry. Overall, 1085 proteins were identified and longitudinally quantified. Principal component analysis (PCA) showed the appearance of the pathology onset between twelve and fifteen months, correlating with sharp amyloid plaque accumulation within the same ages. Cluster analysis followed by protein-protein interaction analysis revealed an age-dependent decrease in mitochondrial protein expression. We identified 57 significantly affected mitochondrial proteins, several of which have been reported to alter expression in neurological diseases. We also found ten proteins that are upregulated early in the amyloid driven pathology progression with high confidence, some of which are directly involved in the onset of mitochondrial apoptosis and may represent potential markers for use in human neurological diseases prognosis. Our results further contribute to identifying common pathological pathways involved in both aging and progressive neurodegenerative disorders enhancing the understanding of disease pathogenesis.

  • 24.
    Sjödin, Marcus O.D.
    et al.
    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.
    Mining ventricular cerebrospinal fluid from patients with traumatic brain injury using hexapeptide ligand libraries to search for trauma biomarkers2010In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 878, no 22, p. 2003-2012Article in journal (Refereed)
    Abstract [en]

    Traumatic brain injury (TBI) is an acute event resulting from external force to the brain and is a major cause of death and disability associated with high health care costs in the western world. Additional injuries, originating from the secondary molecular events after the initial intensive care, may be limited by the use of objective biomarkers to provide the best treatment and patient prediction outcome. In this study, hexapeptide ligand libraries (HLL) have been used for the enrichment of suggested protein biomarkers for TBI in cerebrospinal fluid (CSF). HLL have the potential to enrich low abundant proteins and simultaneously reduce the high abundant proteins, rendering a sample with significantly reduced dynamic range. The CSF proteome from two TBI inflicted patients have been extensively mapped using a large initial sample volume obtained by extraventricular drainage. Shotgun proteomics, in combination with isoelectric focusing (IEF) and nano-LC-MS/MS, identified 339 unique proteins (MudPIT scoring p ≤ 0.05) with a protein overlap of 130 between the patients. As much as 45% of the proteins reported in the literature to be associated with degenerative/regenerative processes occurring after a trauma to the head were identified. Out of the most prominent potential protein biomarkers, such as neuron specific enolase, glial fibrillary acidic protein, myelin basic protein, creatine kinase B-type and S-100β, all except myelin basic protein were detected in the study. This study shows the possibility of using HLL as a tool for screening of low abundant protein biomarkers in human CSF.

  • 25.
    Sjödin, Marcus O.D.
    et al.
    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.
    Kultima, Kim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Artemenko, Konstantin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Comparative study of label and label-free techniques using shotgun proteomics for relative protein quantification2013In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 928, p. 83-92Article in journal (Refereed)
    Abstract [en]

    The analytical performance of three different strategies, iTRAQ (isobaric tag for relative and absolute quantitation), dimethyl labeling (DML) and label free (LF) for relative protein quantification using shotgun proteomics have been evaluated. The methods have been explored using samples containing (i) Bovine proteins in known ratios and (ii) Bovine proteins in known ratios spiked into E.Coli. The latter case mimics the actual conditions in a typical biological sample with a few differentially expressed proteins and a bulk of proteins with unchanged ratios. Additionally, the evaluation was performed on both Q-TOF and LTQ-FTICR mass spectrometers. LF LTQ-FTICR was found to have the highest proteome coverage (94 %) while the highest accuracy based on the artificially regulated proteins was found for DML LTQ-FTICR (54%). A good linearity (r2: 0.61-0.96) was shown for all methods within selected dynamic ranges. All methods were found to consistently underestimate bovine protein ratios when matrix proteins were added. However LF LTQ-FTICR was more tolerant towards a compression effect.  A single peptide was demonstrated to be sufficient for a reliable quantification using iTRAQ. A ranking system utilizing several parameters important for quantitative proteomics demonstrated that the overall performance of the five different methods were; DML LTQ-FTICR > iTRAQ QTOF > LF LTQ-FTICR > DML Q-TOF > LF Q-TOF.

  • 26.
    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.

  • 27.
    Tsybin, Youri O.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Håkansson, Per
    Wetterhall, Magnus
    Markides, Karin E.
    Bergquist, Jonas
    Capillary Electrophoresis and Electron Capture Dissociation Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for Peptide Mixture and Protein Digest Analysis2002In: European journal of mass spectrometry, ISSN 1469-0667, E-ISSN 1751-6838, Vol. 8, no 5, p. 389-395Article in journal (Refereed)
  • 28.
    Ullsten, Sara
    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.
    Wetterhall, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Hardenborg, Emilia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    E. Markides, Karin
    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.
    A polyamine coating for enhanced capillary electrophoresis-electrospray ionization-mass spectrometry of proteins and peptides.2004In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 25, no 13, p. 2090-2099Article in journal (Refereed)
    Abstract [en]

    A procedure for enhanced capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) of proteins is presented. The use of a newly presented capillary coating, PolyE-323, provided fast separations of typically a few minutes with high efficiency, good deactivation, and no bleeding into the mass spectrometer. Capillaries coated with PolyE-323 showed high stability over a range of pH 2-10, and tolerance towards methanol and acetonitrile, two modifiers commonly used in CE-ESI-MS. Due to the speed and simplicity of the coating procedure, the polymeric surface could, if necessary, easily be regenerated. This capability is especially valuable when working with samples of complex matrix, where a capillary surface cleaning step might be desired in order to eliminate possible memory effects. The potential of PolyE-323-coated capillaries in bioanalysis using CE-ESI-MS was demonstrated by analyzing peptides and proteins up to 66 kDa using time of flight (TOF)-MS. Due to the stable, anodal electroosmotic flow generated by the coating, the use of a sheathless ESI interface was enabled, demonstrated in peptide analysis with attomole sensitivity. The fast on-line CE-ESI-TOF system using PolyE-323-coated capillaries provided efficient separation and detection of a large number of peaks in a short time, exemplified by the analysis of a tryptic digest of bovine serum albumin (BSA). The capability of the developed capillary surface coating was demonstrated by the separation of human plasma and cerebrospinal fluid (CSF).

  • 29.
    Undin, Torgny
    et al.
    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.
    Dahlin, Andreas P
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Microsystems Technology.
    Wetterhall, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Competitive Protein Adsorption as Observed and Quantified by - Surface Enzymatic Digestion (oSED) and Mass Spectrometry2012In: 60th ASMS Conference on Mass Spectrometry and Allied Topics, May 20 - 24, Vancouver, Canada, 2012Conference paper (Refereed)
  • 30.
    Wetterhall, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry.
    Electrifying the Molecules of Life: Peptide and Protein Analysis by Capillary Electrophoresis Coupled to Electrospray Ionization Mass Spectrometry2004Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This thesis describes the current status and novel aspects of the analysis of the molecules of life, i.e. peptides and proteins, using capillary electrophoresis (CE) coupled to mass spectrometry (MS) via (sheathless) electrospray ionization (ESI). Early reports of sheathless CE-ESI-MS were plagued by limited lifetimes of the electrospray emitter. In this thesis, two new approaches, the Black Dust and the Black Jack methods, utilizing polymer-embedded graphite instead of noble metals are presented. These emitters have shown improved long-term stability and proven excellent for sheathless electrospray operation. Failure of an emitter is often caused by electrochemical reactions occurring at the emitter-liquid interface. The electrochemical properties of the graphite coated emitters were therefore evaluated by classical electrochemical methods, such as cyclic voltammetry and chronoamperometry. The graphite coated emitters showed excellent electrochemical stability and properties compared to noble metal and polymer configurations.

    Analyte-wall interactions have long been known to cause problems in the CE analysis of biomolecules. This can be circumvented by internal modification of the capillary walls. Additionally, it is of outermost importance to have a stable and sufficiently high electroosmotic flow (EOF) to sustain the electrospray, when using a sheathless approach. New monomer and polymer coatings are presented for rapid and high-efficient CE-ESI-MS separations of peptides and proteins.

    Furthermore, the use of CE-ESI coupled to Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) shows great potential for rapid proteomic probing of human cerebrospinal fluid. The results are comparable with more established techniques, such as liquid chromatography and two-dimensional gel electrophoresis coupled to MS. However, the CE-ESI-FTICRMS analysis has significantly lower sample consumption and faster analysis time compared to the other techniques. The applications and use of CE-ESI-MS is expected to have a bright future with continued growth as current trends of multidimensional hyphenation and microfabricated devices are further developed and explored.

    List of papers
    1. A simple and robust conductive graphite coating for sheathless electrospray emitters used in capillary electrophoresis/mass spectrometry
    Open this publication in new window or tab >>A simple and robust conductive graphite coating for sheathless electrospray emitters used in capillary electrophoresis/mass spectrometry
    Show others...
    2001 (English)In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 15, no 21, p. 1997-2000Article in journal (Refereed) Published
    Abstract [en]

    A graphite-polyimide mixture was used as a conductive coating for sheathless electrospray emitters. The coating procedure described is simple and inexpensive compared to previously described methods. An investigation of the stability of the conductive coating carried out by electrochemical methods revealed good performances during oxidative stress. In addition, no decrease in emitter performance was seen during continuous electrospray in the positive electrospray mode for two weeks. Fast capillary electrophoresis with attomole sensitivity demonstrated the excellent performance of the described sheathless interface when used in conjunction with an orthogonal time-of-flight mass spectrometer. The overall simplicity, stability and low cost of this type of sheathless emitter make the described approach highly suitable for any on-column coupling of low flow rate separation techniques to a mass spectrometer.

    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-91884 (URN)10.1002/rcm.466 (DOI)
    Available from: 2004-05-25 Created: 2004-05-25 Last updated: 2017-12-14Bibliographically approved
    2. A conductive polymeric material used for nanospray needle and low-flow sheathless electrospray ionization applications
    Open this publication in new window or tab >>A conductive polymeric material used for nanospray needle and low-flow sheathless electrospray ionization applications
    2002 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 74, no 1, p. 239-245Article in journal (Refereed) Published
    Abstract [en]

    A conductive polypropylene/graphite mixture is used for the production of polymeric nanospray needle emitters and as a coating on fused-silica capillaries that are used for sheathless electrospray ionization (ESI). The described production of these polymeric nanospray needle emitters and sheathless ESI contacts is exceptionally easy and at a very low cost. The described polymeric nanospray emitters have shown excellent features regarding their chemical inertness and spray performance. The long-term stability of the nanospray needles exceeds 24 h of continuous use. Furthermore, the resistance to electrical discharges, which is one of the factors that often limits the lifetime of metal coated tips, has proven to be outstanding. A voltage of up to 5 kV could be applied without loss of spray performance. The use of polypropylene emitters offers a number of desirable features, as compared to silica based emitters. Among these features are mechanical flexibility and simplified regeneration of the nanospray needle. Continuous nanospray of peptides and proteins in conjunction with orthogonal time-of-flight mass spectrometry are shown with signal relative standard deviations of 5%. In addition, the polypropylene/graphite mixture has also been applied as the conductive contact for sheathless ESI in fast capillary electrophoresis separations.

    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-91885 (URN)10.1021/ac010748f (DOI)
    Available from: 2004-05-25 Created: 2004-05-25 Last updated: 2017-12-14Bibliographically approved
    3. A comparison of the electrochemical stabilities of metal, polymer and graphite coated nanospray emitters
    Open this publication in new window or tab >>A comparison of the electrochemical stabilities of metal, polymer and graphite coated nanospray emitters
    Show others...
    2003 (English)In: The Analyst, ISSN 0003-2654, Vol. 128, no 6, p. 728-733Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-91665 (URN)
    Available from: 2004-04-20 Created: 2004-04-20 Last updated: 2011-05-09
    4. Monomer surface modifications for rapid peptide analysis by capillary electrphoresis and capillary electrochromatography coupled to electrospray ionization mass spectrometry
    Open this publication in new window or tab >>Monomer surface modifications for rapid peptide analysis by capillary electrphoresis and capillary electrochromatography coupled to electrospray ionization mass spectrometry
    2004 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 25, no 6, p. 809-816Article in journal (Refereed) Published
    Abstract [en]

    In this study, positively charged alkylaminosilyl monomers were used to modify the inner surface of fused silica capillaries, which subsequently were employed in capillary electrophoresis (CE) and capillary electrochromatography (CEC). The obtained surfaces yield a reversed electroosmotic flow (EOF) and have varying carbon chain lengths, that interact with the analytes and give chromatographic retention. The coating procedure is very simple and fast. The performance of the modified capillaries was evaluated regarding pH influence on EOF and chromatographic interactions. The experiments were conducted with UV and mass spectrometry (MS) and applied to the separation of various neuropeptides. The derivatized surfaces showed a linear (R2 ∼0.99) pH dependence with isoelectric points (pI) at 8.6–8.8. Rapid separations of peptide standards and a protein digest with efficiencies as high as 5×105 plates/m were performed.

    Keywords
    Capillary electrochromatography, Capillary electrophoresis, Mass spectrometry, Peptide, Surface modification
    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-94824 (URN)10.1002/elps.200305719 (DOI)
    Available from: 2006-09-15 Created: 2006-09-15 Last updated: 2017-12-14Bibliographically approved
    5. A polyamine coating for enhanced capillary electrophoresis-electrospray ionization-mass spectrometry of proteins and peptides.
    Open this publication in new window or tab >>A polyamine coating for enhanced capillary electrophoresis-electrospray ionization-mass spectrometry of proteins and peptides.
    Show others...
    2004 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 25, no 13, p. 2090-2099Article in journal (Refereed) Published
    Abstract [en]

    A procedure for enhanced capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) of proteins is presented. The use of a newly presented capillary coating, PolyE-323, provided fast separations of typically a few minutes with high efficiency, good deactivation, and no bleeding into the mass spectrometer. Capillaries coated with PolyE-323 showed high stability over a range of pH 2-10, and tolerance towards methanol and acetonitrile, two modifiers commonly used in CE-ESI-MS. Due to the speed and simplicity of the coating procedure, the polymeric surface could, if necessary, easily be regenerated. This capability is especially valuable when working with samples of complex matrix, where a capillary surface cleaning step might be desired in order to eliminate possible memory effects. The potential of PolyE-323-coated capillaries in bioanalysis using CE-ESI-MS was demonstrated by analyzing peptides and proteins up to 66 kDa using time of flight (TOF)-MS. Due to the stable, anodal electroosmotic flow generated by the coating, the use of a sheathless ESI interface was enabled, demonstrated in peptide analysis with attomole sensitivity. The fast on-line CE-ESI-TOF system using PolyE-323-coated capillaries provided efficient separation and detection of a large number of peaks in a short time, exemplified by the analysis of a tryptic digest of bovine serum albumin (BSA). The capability of the developed capillary surface coating was demonstrated by the separation of human plasma and cerebrospinal fluid (CSF).

    Keywords
    Capillary electrophoresis, mass spectrometry, peptides, polyamine coating, proteins
    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-98065 (URN)10.1002/elps.200305787 (DOI)000222890600019 ()15237410 (PubMedID)
    Available from: 2009-02-20 Created: 2009-02-06 Last updated: 2017-12-13Bibliographically approved
    6. Rapid analysis of tryptically digested cerebrospinal fluid using capillary electrophoresis-electrospray ionization-Fourier transform ion cyclotron resonance-mass spectrometry
    Open this publication in new window or tab >>Rapid analysis of tryptically digested cerebrospinal fluid using capillary electrophoresis-electrospray ionization-Fourier transform ion cyclotron resonance-mass spectrometry
    Show others...
    2002 (English)In: Journal of Proteome Research, ISSN 1535-3893, Vol. 1, no 4, p. 361-366Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-91668 (URN)
    Available from: 2004-04-20 Created: 2004-04-20 Last updated: 2011-05-09
  • 31.
    Wetterhall, Magnus
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry.
    Klett, Oliver
    Markides, Karin E.
    Nyholm, Leif
    Bergquist, Jonas
    A comparison of the electrochemical stabilities of metal, polymer and graphite coated nanospray emitters2003In: The Analyst, ISSN 0003-2654, Vol. 128, no 6, p. 728-733Article in journal (Refereed)
  • 32.
    Wetterhall, Magnus
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Nilsson, Stefan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Markides, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    A conductive polymeric material used for nanospray needle and low-flow sheathless electrospray ionization applications2002In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 74, no 1, p. 239-245Article in journal (Refereed)
    Abstract [en]

    A conductive polypropylene/graphite mixture is used for the production of polymeric nanospray needle emitters and as a coating on fused-silica capillaries that are used for sheathless electrospray ionization (ESI). The described production of these polymeric nanospray needle emitters and sheathless ESI contacts is exceptionally easy and at a very low cost. The described polymeric nanospray emitters have shown excellent features regarding their chemical inertness and spray performance. The long-term stability of the nanospray needles exceeds 24 h of continuous use. Furthermore, the resistance to electrical discharges, which is one of the factors that often limits the lifetime of metal coated tips, has proven to be outstanding. A voltage of up to 5 kV could be applied without loss of spray performance. The use of polypropylene emitters offers a number of desirable features, as compared to silica based emitters. Among these features are mechanical flexibility and simplified regeneration of the nanospray needle. Continuous nanospray of peptides and proteins in conjunction with orthogonal time-of-flight mass spectrometry are shown with signal relative standard deviations of 5%. In addition, the polypropylene/graphite mixture has also been applied as the conductive contact for sheathless ESI in fast capillary electrophoresis separations.

  • 33.
    Wetterhall, Magnus
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry.
    Palmblad, Magnus
    Håkansson, Per
    Markides, Karin E.
    Bergquist, Jonas
    Rapid analysis of tryptically digested cerebrospinal fluid using capillary electrophoresis-electrospray ionization-Fourier transform ion cyclotron resonance-mass spectrometry2002In: Journal of Proteome Research, ISSN 1535-3893, Vol. 1, no 4, p. 361-366Article in journal (Refereed)
  • 34.
    Wetterhall, Magnus
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Shevchenko, Ganna
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Artemenko, Konstantin A
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Sjödin, Marcus O.D.
    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.
    Analysis of membrane and hydrophilic proteins simultaneously derived from the mouse brain using cloud-point extraction2011In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 400, no 9, p. 2827-2836Article in journal (Refereed)
    Abstract [en]

    In this study, a temperature-induced phase fractionation known as cloud-point extraction (CPE) with the non-ionic surfactant Triton X-114 was used to simultaneously extract, concentrate, and fractionate hydrophobic and hydrophilic proteins from mouse brain tissue. Two bottom-up proteomic techniques were used to comprehensively identify the extracted proteins. The first "shotgun"-based approach included tryptic digestion of the proteins followed by reversed-phase nanoliquid chromatography (RP-nanoLC) in combination with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). In the second approach, the extracted intact proteins were first separated by one-dimensional (1D) gel electrophoresis and then in-gel digested with trypsin and analyzed with nanoLC-MS/MS. In total, 1,825 proteins were unambiguously identified and the percentage of membrane proteins was 26% which is at the reported genome expression levels of 20-30%. The protein overlap between the two approaches was high. The majority (77%) of the identifications in the first approach was also found by the second method. The protein overlap between the CPE-extracted hydrophilic and hydrophobic fractions was rather small (16-23%) for both methods, which indicates a good phase separation. A quantitative evaluation of the CPE with iTRAQ labeling and nanoLC-ESI-MS/MS analysis gave iTRAQ ratios at the expected levels and an overall variation of the entire method at 17-31%. The results indicate very reproducible sample preparation and analysis methods that readily can be applied on large-scale sample sets.

  • 35.
    Wetterhall, Magnus
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Sjödin, Marcus O D
    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.
    Hillered, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Neurosurgery.
    Hjort, Klas
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Microsystems Technology.
    Dahlin, Andreas P
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Microsystems Technology.
    Mapping the protein distribution within a microdialysis sampling system by on-surface enzymatic digestion in combination with mass spectrometry2012In: Monitoring Molecules in Neuroscience: 14th International Conference, September 16 – 20, London, U.K., 2012Conference paper (Refereed)
  • 36.
    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%.

  • 37.
    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)
  • 38.
    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.

  • 39.
    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.

1 - 39 of 39
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