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  • 1.
    Andersson, Per Ola
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Applied Materials Sciences. FOI Swedish Def Res Agcy, CBRN Def & Secur, S-90182 Umea, Sweden.; Mol Fingerprint Sweden AB, Eksatravagen 130, S-75655 Uppsala, Sweden.
    Viberg, Pernilla
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Applied Materials Sciences.
    Forsberg, Pontus
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Applied Materials Sciences.
    Nikolajeff, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Applied Materials Sciences. Mol Fingerprint Sweden AB, Eksatravagen 130, S-75655 Uppsala, Sweden.
    Österlund, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Solid State Physics. Mol Fingerprint Sweden AB, Eksatravagen 130, S-75655 Uppsala, Sweden.
    Karlsson, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Applied Materials Sciences. Mol Fingerprint Sweden AB, Eksatravagen 130, S-75655 Uppsala, Sweden.
    Nanocrystalline diamond sensor targeted for selective CRP detection: An ATR-FTIR spectroscopy study2016In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 408, no 14, p. 3675-3680Article in journal (Refereed)
    Abstract [en]

    Protein immobilization on functionalized fluorine- terminated nanocrystalline (NCD) films was studied by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy using an immobilization protocol developed to specifically bind C-reactive protein (CRP). Using an ATR- FTIR spectroscopy method employing a force-controlled anvil-type configuration, three critical steps of the ex situ CRP immobilization were analyzed. First, the NCD surface was passivated by deposition of a copolymer layer consisting of polyethylene oxide and polypropylene oxide. Second, a synthetic modified polypeptide binder with high affinity to CRP was covalently attached to the polymeric film. Third, CRP dissolved in aqueous buffer in concentrations of 10–20 μg/ mL was added on the functionalized NCD surface. Both the amide I and II bands, due to the polypeptide binder and CRP, were clearly observed in ATR-FTIR spectra. CRP amide I bands were extracted from difference spectra and yielded bands that agreed well with the reported amide I band of free (non-bonded) CRP in solution. Thus, our results show that CRP retains its secondary structure when it is attached to the polypeptide binders. Compared to previous IR studies of CRP in solution, about 200 times lower concentration was applied in the present study. 

  • 2.
    Baltzer, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry.
    Crossing borders to bind proteins-a new concept in protein recognition based on the conjugation of small organic molecules or short peptides to polypeptides from a designed set2011In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 400, no 6, p. 1653-1664Article, review/survey (Refereed)
    Abstract [en]

    A new concept for protein recognition and binding is highlighted. The conjugation of small organic molecules or short peptides to polypeptides from a designed set provides binder molecules that bind proteins with high affinities, and with selectivities that are equal to those of antibodies. The small organic molecules or peptides need to bind the protein targets but only with modest affinities and selectivities, because conjugation to the polypeptides results in molecules with dramatically improved binder performance. The polypeptides are selected from a set of only sixteen sequences designed to bind, in principle, any protein. The small number of polypeptides used to prepare high-affinity binders contrasts sharply with the huge libraries used in binder technologies based on selection or immunization. Also, unlike antibodies and engineered proteins, the polypeptides have unordered three-dimensional structures and adapt to the proteins to which they bind. Binder molecules for the C-reactive protein, human carbonic anhydrase II, acetylcholine esterase, thymidine kinase 1, phosphorylated proteins, the D-dimer, and a number of antibodies are used as examples to demonstrate that affinities are achieved that are higher than those of the small molecules or peptides by as much as four orders of magnitude. Evaluation by pull-down experiments and ELISA-based tests in human serum show selectivities to be equal to those of antibodies. Small organic molecules and peptides are readily available from pools of endogenous ligands, enzyme substrates, inhibitors or products, from screened small molecule libraries, from phage display, and from mRNA display. The technology is an alternative to established binder concepts for applications in drug development, diagnostics, medical imaging, and protein separation.

  • 3.
    Barclay, Victoria K. H.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Tyrefors, Niklas L
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Johansson, I. Monika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Pettersson, Curt E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Acidic transformation of nordiazepam can affect recovery estimate during trace analysis of diazepam and nordiazepam in environmental water samples by liquid chromatography-tandem mass spectrometry2019In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 411, no 17, p. 3919-3928Article in journal (Refereed)
    Abstract [en]

    In this study, a special interest was focused on the stability of diazepam and nordiazepam in aqueous samples at acidic and neutral pH. The aim of the study was to isolate and illustrate one of the many possible sources of error that can be encountered when developing and validating analytical methods. This can be of particular importance when developing multi-analyte methods where there is limited time to scrutinize the behavior of each analyte. A method was developed for the analysis of the benzodiazepines diazepam and nordiazepam in treated wastewater. The samples were extracted by solid phase extraction, using SPEC C18AR cartridges, and analyzed by the use of liquid chromatography, with a C18 stationary phase, coupled to tandem mass spectrometry. Environmental water samples are often acidified during storage to reduce the microbial degradation of the target compounds and to preserve the sample. In some cases, the samples are acidified before extraction. In this study, it was found that a chemical equilibrium between nordiazepam and a transformation product could cause inaccurately high extraction recovery values when the samples were stored at low sample pH. The stability of nordiazepam was shown to be low at pH3. Within 12days, 20% of the initial concentration of nordiazepam was transformed. Interestingly, the transformed nordiazepam was shown to be regenerated and reformed to nordiazepam during sample handling. At a sample pH of 7, diazepam and nordiazepam were stable for 12days. It was concluded that great care must be taken when acidifying water samples containing nordiazepam during storage or extraction. The storage and the extraction should be conducted at neutral pH if no internal standard is used to compensate for degradation and conversion of nordiazepam. The developed method was validated in treated wastewater and applied for the quantification of diazepam and nordiazepam in treated wastewater samples.

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  • 4.
    Bergman, Hilde-Marlene
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Lindfors, Lina
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Palm, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Kihlberg, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Lanekoff, Ingela
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Metabolite aberrations in early diabetes detected in rat kidney using mass spectrometry imaging2019In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 411, no 13, p. 2809-2816Article in journal (Refereed)
    Abstract [en]

    Diabetic kidney disease is a serious complication of diabetes that can ultimately lead to end-stage renal disease. The pathogenesis of diabetic kidney disease is complex, and fundamental research is still required to provide a better understanding of the driving forces behind it. We report regional metabolic aberrations from an untargeted mass spectrometry imaging study of kidney tissue using an insulinopenic rat model of diabetes. Diabetes was induced by intravenous injection of streptozotocin, and kidneys were harvested 2weeks thereafter. Imaging was performed using nanospray desorption electrospray ionization connected to a high-mass-resolving mass spectrometer. No histopathological changes were observed in the kidney sections; however, mass spectrometry imaging revealed a significant increase in several 18-carbon unsaturated non-esterified fatty acid species and monoacylglycerols. Notably, these 18-carbon acyl chains were also constituents of several increased diacylglycerol species. In addition, a number of short- and long-chain acylcarnitines were found to be accumulated while several amino acids were depleted. This study presents unique regional metabolic data indicating a dysregulated energy metabolism in renal mitochondria as an early response to streptozotocin-induced type I diabetes.

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  • 5.
    Bergman, Nina
    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. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Ingvar Eidhammer, Harald Barsnes, Geir Egil Eide, and Lennart Martens:: Computational and statistical methods for protein quantification by mass spectrometry2014In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 406, no 6, p. 1575-1576Article, book review (Refereed)
  • 6.
    Bergman, Nina
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Shevchenko, Denys
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Approaches for the analysis of low molecular weight compounds with laser desorption/ionization techniques and mass spectrometry2014In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 406, no 1, p. 49-61Article, review/survey (Refereed)
    Abstract [en]

    This review summarizes various approaches for the analysis of low molecular weight (LMW) compounds by different laser desorption/ionization mass spectrometry techniques (LDI-MS). It is common to use an agent to assist the ionization, and small molecules are normally difficult to analyze by, e.g., matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) using the common matrices available today, because the latter are generally small organic compounds themselves. This often results in severe suppression of analyte peaks, or interference of the matrix and analyte signals in the low mass region. However, intrinsic properties of several LDI techniques such as high sensitivity, low sample consumption, high tolerance towards salts and solid particles, and rapid analysis have stimulated scientists to develop methods to circumvent matrix-related issues in the analysis of LMW molecules. Recent developments within this field as well as historical considerations and future prospects are presented in this review.

  • 7.
    Bergquist, Jonas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Emmer, Åsa
    Analytical Chemistry, Division of Applied Physical Chemistry, Department of Chemistry, KTH Royal Institute of Technology, Teknikringen 36, SE-10044, Stockholm, Sweden..
    Farbrot, Anne
    Department of Occupational and Environmental Health, Sahlgren’s University Hospital, P.O. Box 414, SE-40530, Gothenburg, Sweden..
    Turner, Charlotta
    Department of Chemistry, Centre for Analysis and Synthesis, Lund University, P.O. Box 124, SE-22100, Lund, Sweden..
    Research and education in analytical chemistry: Industrial and academic perspectives from a survey conducted in Sweden2023In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 415, p. 2151-2161Article in journal (Refereed)
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  • 8.
    Bergquist, Jonas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Turner, Charlotta
    Lund Univ, Dept Chem, Ctr Anal & Synth, Lund, Sweden..
    Analytical chemistry for a sustainable society - trends and implications2018In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 410, no 14, p. 3235-3237Article in journal (Other academic)
  • 9. Bjornstad, Kristian
    et al.
    Åberg, Annica Tevell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Kalb, Suzanne R.
    Wang, Dongxia
    Barr, John R.
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Validation of the Endopep-MS method for qualitative detection of active botulinum neurotoxins in human and chicken serum2014In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 406, no 28, p. 7149-7161Article in journal (Refereed)
    Abstract [en]

    Botulinum neurotoxins (BoNTs) are highly toxic proteases produced by anaerobic bacteria. Traditionally, a mouse bioassay (MBA) has been used for detection of BoNTs, but for a long time, laboratories have worked with alternative methods for their detection. One of the most promising in vitro methods is a combination of an enzymatic and mass spectrometric assay called Endopep-MS. However, no comprehensive validation of the method has been presented. The main purpose of this work was to perform a validation for the qualitative analysis of BoNT-A, B, C, C/D, D, D/C, and F in serum. The limit of detection (LOD), selectivity, precision, stability in matrix and solution, and correlation with the MBA were evaluated. The LOD was equal to or even better than that of the MBA for BoNT-A, B, D/C, E, and F. Furthermore, Endopep-MS was for the first time successfully used to differentiate between BoNT-C and D and their mosaics C/D and D/C by different combinations of antibodies and target peptides. In addition, sequential antibody capture was presented as a new way to multiplex the method when only a small sample volume is available. In the comparison with the MBA, all the samples analyzed were positive for BoNT-C/D with both methods. These results indicate that the Endopep-MS method is a valid alternative to the MBA as the gold standard for BoNT detection based on its sensitivity, selectivity, and speed and that it does not require experimental animals.

  • 10.
    Chu, Jiangtao
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Microsystems Technology.
    Koudriavtsev, Vitali
    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.
    Dahlin, Andreas P
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Microsystems Technology.
    Fluorescence imaging of macromolecule transport in high molecular weight cut-off microdialysis2014In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 406, no 29, p. 7601-7609Article in journal (Refereed)
    Abstract [en]

    When microdialysis (MD) membrane exceeds molecular weight cut-off (MWCO) of 100 kDa, the fluid mechanics are in the ultrafiltration regime. Consequently, fluidic mass transport of macromolecules in the perfusate over the membrane may reduce the biological relevance of the sampling and cause an inflammatory response in the test subject. Therefore, a method to investigate the molecular transport of high MWCO MD is presented. An in vitro test chamber was fabricated to facilitate the fluorescent imaging of the MD sampling process, using fluoresceinylisothiocyanate (FITC) dextran and fluorescence microscopy. Qualitative studies on dextran behavior inside and outside the membrane were performed. Semiquantitative results showed clear dextran leakage from both 40 and 250 kDa dextran when 100 kDa MWCO membranes were used. Dextran 40 kDa leaked out with an order of magnitude higher concentration and the leakage pattern resembled more of a convective flow pattern compared with dextran 250 kDa, where the leakage pattern was more diffusion based. No leakage was observed when dextran 500 kDa was used as a colloid osmotic agent. The results in this study suggest that fluorescence imaging could be used as a method for qualitative and semiquantitative molecular transport and fluid dynamics studies of MD membranes and other hollow fiber catheter membranes.

  • 11.
    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.

  • 12.
    Enmark, Martin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Farmakognosi. Karlstad Univ, Dept Engn & Chem Sci, S-65188 Karlstad, Sweden.
    Bagge, Joakim
    Karlstad Univ, Dept Engn & Chem Sci, S-65188 Karlstad, Sweden.
    Samuelsson, Jorgen
    Karlstad Univ, Dept Engn & Chem Sci, S-65188 Karlstad, Sweden.
    Thunberg, Linda
    AstraZeneca, BioPharmaceut R&D, Pharmaceut Sci, Early Chem Dev, S-43183 Mölndal, Sweden.
    Ornskov, Eivor
    AstraZeneca, BioPharmaceut R&D, Pharmaceut Sci, Adv Drug Delivery, S-43183 Mölndal, Sweden.
    Leek, Hanna
    AstraZeneca, BioPharmaceut R&D, Pharmaceut Sci, Early Chem Dev, S-43183 Mölndal, Sweden.
    Lime, Fredrik
    Nouryon, Separat Prod, S-44580 Bohus, Sweden.
    Fornstedt, Torgny
    Karlstad Univ, Dept Engn & Chem Sci, S-65188 Karlstad, Sweden.
    Analytical and preparative separation of phosphorothioated oligonucleotides: columns and ion-pair reagents2020In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 412, no 2, p. 299-309Article in journal (Refereed)
    Abstract [en]

    Oligonucleotide drugs represent an emerging area in the pharmaceutical industry. Solid-phase synthesis generates many structurally closely related impurities, making efficient separation systems for purification and analysis a key challenge during pharmaceutical drug development. To increase the fundamental understanding of the important preparative separation step, mass-overloaded injections of a fully phosphorothioated 16mer, i.e., deoxythymidine oligonucleotide, were performed on a C18 and a phenyl column. The narrowest elution profiles were obtained using the phenyl column, and the 16mer could be collected with high purity and yield on both columns. The most likely contribution to the successful purification was the quantifiable displacement of the early-eluting shortmers on both columns. In addition, the phenyl column displayed better separation of later-eluting impurities, such as the 17mer impurity. The mass-overloaded injections resulted in classical Langmuirian elution profiles on all columns, provided the concentration of the ion-pairing reagent in the eluent was sufficiently high. Two additional column chemistries, C4 and C8, were also investigated in terms of their selectivity and elution profile characteristics for the separation of 520mers fully phosphorothioated deoxythymidine oligonucleotides. When using triethylamine as ion-pairing reagent to separate phosphorothioated oligonucleotides, we observed peak broadening caused by the partial separation of diastereomers, predominantly seen on the C4 and C18 columns. When using the ion-pair reagent tributylamine, to suppress diastereomer separation, the greatest selectivity was found using the phenyl column followed by C18. The present results will be useful when designing and optimizing efficient preparative separations of synthetic oligonucleotides.

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  • 13.
    Enmark, Martin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Farmakognosi. Karlstad Univ, Dept Engn & Chem Sci, S-65188 Karlstad, Sweden.
    Rova, Maria
    Karlstad Univ, Dept Engn & Chem Sci, S-65188 Karlstad, Sweden.
    Samuelsson, Jorgen
    Karlstad Univ, Dept Engn & Chem Sci, S-65188 Karlstad, Sweden.
    Ornskov, Eivor
    AstraZeneca, IMED Biotech Unit, Pharmaceut Sci, Adv Drug Delivery, S-43183 Gothenburg, Sweden.
    Schweikart, Fritz
    AstraZeneca, IMED Biotech Unit, Pharmaceut Sci, Adv Drug Delivery, S-43183 Gothenburg, Sweden.
    Fornstedt, Torgny
    Karlstad Univ, Dept Engn & Chem Sci, S-65188 Karlstad, Sweden.
    Investigation of factors influencing the separation of diastereomers of phosphorothioated oligonucleotides2019In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 411, no 15, p. 3383-3394Article in journal (Refereed)
    Abstract [en]

    This study presents a systematic investigation of factors influencing the chromatographic separation of diastereomers of phosphorothioated pentameric oligonucleotides as model solutes. Separation was carried out under ion-pairing conditions using an XBridge C-18 column. For oligonucleotides with a single sulfur substitution, the diastereomer selectivity was found to increase with decreasing carbon chain length of the tertiary alkylamine used as an ion-pair reagent. Using an ion-pair reagent with high selectivity for diastereomers, triethylammonium, it was found the selectivity increased with decreased ion-pair concentration and shallower gradient slope. Selectivity was also demonstrated to be dependent on the position of the modified linkage. Substitutions at the center of the pentamer resulted in higher diastereomer selectivity compared to substitutions at either end. For mono-substituted oligonucleotides, the retention order and stereo configuration were consistently found to be correlated, with Rp followed by Sp, regardless of which linkage was modified. The type of nucleobase greatly affects the observed selectivity. A pentamer of cytosine has about twice the diastereomer selectivity of that of thymine. When investigating the retention of various oligonucleotides eluted using tributylammonium as the ion-pairing reagent, no diastereomer selectivity could be observed. However, retention was found to be dependent on both the degree and position of sulfur substitution as well as on the nucleobase. When analyzing fractions collected in the front and tail of overloaded injections, a significant difference was found in the ratio between Rp and Sp diastereomers, indicating that the peak broadening observed when using tributylammonium could be explained by partial diastereomer separation.

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  • 14.
    Fromell, Karin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology.
    Forsberg, Pontus
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Microsystems Technology.
    Karlsson, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Microsystems Technology.
    Larsson, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Inorganic Chemistry.
    Nikolajeff, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Microsystems Technology.
    Baltzer, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Physical Organic Chemistry.
    Designed protein binders in combination with nanocrystalline diamond for use in high-sensitivity biosensors2012In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 404, no 6-7, p. 1643-1651Article in journal (Refereed)
    Abstract [en]

    A platform for diagnostic applications showing signal-to-noise ratios that by far surpass those of traditional bioanalytical test formats has been developed. It combines the properties of modified nanocrystalline diamond (NCD) surfaces and those of polyethylene oxide and polypropylene oxide based block copolymers for surface passivation and binder conjugation with a new class of synthetic binders for proteins. The NCD surfaces were fluorine-, hydrogen-, or oxygen-terminated prior to further biofunctionalization and the surface composition was characterized by X-ray photoelectron spectroscopy. In a proof of principle demonstration targeting the C-reactive protein, an ELISA carried out using an F-terminated diamond surface showed a signal-to-noise ratio of 3,900 which compares well to the signal-to-noise of 89 obtained in an antibody-based ELISA on a polystyrene microtiter plate, a standard test format used in most life science laboratories today. The increase in signal-to-noise ratio is to a large extent the result of extremely efficient passivation of the diamond surface. The results suggest that significant improvements can be obtained in standardized test formats using new materials in combination with new types of chemical coatings and receptor molecules.

  • 15. Guddat, S.
    et al.
    Fusshoeller, G.
    Beuck, S.
    Thomas, A.
    Geyer, H.
    Rydevik, Axel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Lagojda, A.
    Schaenzer, W.
    Thevis, M.
    Synthesis, characterization, and detection of new oxandrolone metabolites as long-term markers in sports drug testing2013In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 405, no 25, p. 8285-8294Article in journal (Refereed)
    Abstract [en]

    The discovery and implementation of the long-term metabolite of metandienone, namely 17 beta-hydroxymethyl-17 alpha-methyl-18-norandrost-1,4,13-trien-3-one, to doping control resulted in hundreds of positive metandienone findings worldwide and impressively demonstrated that prolonged detection periods significantly increase the effectiveness of sports drug testing. For oxandrolone and other 17-methyl steroids, analogs of this metabolite have already been described, but comprehensive characterization and pharmacokinetic data are still missing. In this report, the synthesis of the two epimeric oxandrolone metabolites-17 beta-hydroxymethyl-17 alpha-methyl-18-nor-2-oxa-5 alpha-androsta-13-en-3-one and 17 alpha-hydroxymethyl-17 beta-methyl-18-nor-2-oxa-5 alpha-androsta-13-en-3-one-using a fungus (Cunninghamella elegans) based protocol is presented. The reference material was fully characterized by liquid chromatography nuclear magnetic resonance spectroscopy and high resolution/high accuracy mass spectrometry. To ensure a specific and sensitive detection in athlete's urine, different analytical approaches were followed, such as liquid chromatography-tandem mass spectrometry (QqQ and Q-Orbitrap) and gas chromatography-tandem mass spectrometry, in order to detect and identify the new target analytes. The applied methods have demonstrated good specificity and no significant matrix interferences. Linearity (R (2) > 0.99) was tested, and precise results were obtained for the detection of the analytes (coefficient of variation < 20 %). Limits of detection (S/N) for confirmatory and screening analysis were estimated at 1 and 2 ng/mL of urine, respectively. The assay was applied to oxandrolone post-administration samples to obtain data on the excretion of the different oxandrolone metabolites. The studied specimens demonstrated significantly longer detection periods (up to 18 days) for the new oxandrolone metabolites compared to commonly targeted metabolites such as epioxandrolone or 18-nor-oxandrolone, presenting a promising approach to improve the fight against doping.

  • 16.
    Hanrieder, Jörg
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Wicher, Grzegorz
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Andersson, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Fex-Svenningsen, Asa
    Institute of Medical Biology, Neurobiology Research, University of Southern Denmark.
    MALDI mass spectrometry based molecular phenotyping of CNS glial cells for prediction in mammalian brain tissue2011In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 401, no 1, p. 135-147Article in journal (Refereed)
    Abstract [en]

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

  • 17. Jarmalaviciene, Reda
    et al.
    Kornysova, Olga
    Bendokas, Vidmantas
    Westerlund, Douglas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Buszewski, Boguslaw
    Maruska, Audrius
    Restricted-access media development for direct analysis of drugs in biofluids using capillary liquid chromatography2008In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 391, no 6, p. 2323-2328Article in journal (Refereed)
    Abstract [en]

    In analytical sciences the design of novel materials and stationary phases for the sample preparation and separation of analytes from biological fluids is needed. In this work we present different strategies for modification of stationary phases to produce tailored solutions for the analytical problem. In this context a novel shielded polymeric reversed-phase monolithic material was prepared in the presence of different numbers of reactive groups and concentrations of the coating polymer. Chromatographic experiments were performed using benzoic acid propyl ester in order to characterize the hydrophobicity and efficiency of the different restricted-access continuous beds prepared. Inverse size-exclusion chromatography was used for investigation of the pore structure properties of the beds. Capillary columns were applied for nanochromatography of biological fluids containing a mixture of nitrazepamum and medazepamum.

  • 18. Kierspel, Thomas
    et al.
    Kadek, Alan
    Barran, Perdita
    Bellina, Bruno
    Bijedic N, Adi
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy.
    Brodmerkel, Maxim N.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Commandeur, Jan
    Caleman, Carl
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Chemical and Bio-Molecular Physics. Centre for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, Notkestraße 85, E22607, Hamburg, Germany.
    Damjanović, Tomislav
    Dawod, Ibrahim
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Chemical and Bio-Molecular Physics. European XFEL, Holzkoppel 4, 22869, Schenefeld, Germany.
    De Santis, Emiliano
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Lekkas, Alexandros
    Lorenzen, Kristina
    López Morillo, Luis
    Mandl, Thomas
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Chemical and Bio-Molecular Physics. University of Applied Sciences Technikum Wien, Höchstädtpl. 6, 1200, Vienna, Austria.
    Marklund, Erik G.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Papanastasiou, Dimitris
    Ramakers, Lennart A. I.
    Schweikhard, Lutz
    Simke, Florian
    Sinelnikova, Anna
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Materials Theory.
    Smyrnakis, Athanasios
    Timneanu, Nicusor
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Chemical and Bio-Molecular Physics.
    Uetrecht, Charlotte
    Coherent diffractive imaging of proteins and viral capsids: simulating MS SPIDOC2023In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 415, no 18 SI, p. 4209-4220Article in journal (Refereed)
    Abstract [en]

    MS SPIDOC is a novel sample delivery system designed for single (isolated) particle imaging at X-ray Free-Electron Lasers that is adaptable towards most large-scale facility beamlines. Biological samples can range from small proteins to MDa particles. Following nano-electrospray ionization, ionic samples can be m/z-filtered and structurally separated before being oriented at the interaction zone. Here, we present the simulation package developed alongside this prototype. The first part describes how the front-to-end ion trajectory simulations have been conducted. Highlighted is a quadrant lens; a simple but efficient device that steers the ion beam within the vicinity of the strong DC orientation field in the interaction zone to ensure spatial overlap with the X-rays. The second part focuses on protein orientation and discusses its potential with respect to diffractive imaging methods. Last, coherent diffractive imaging of prototypical T = 1 and T = 3 norovirus capsids is shown. We use realistic experimental parameters from the SPB/SFX instrument at the European XFEL to demonstrate that low-resolution diffractive imaging data (q < 0.3 nm−1) can be collected with only a few X-ray pulses. Such low-resolution data are sufficient to distinguish between both symmetries of the capsids, allowing to probe low abundant species in a beam if MS SPIDOC is used as sample delivery.

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  • 19.
    Källsten, Malin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Zhao, Hongxing
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Konzer, Anne
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lind, Sara Bergström
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    A comparative study of phosphopeptide-selective techniques for a sub-proteome of a complex biological sample.2016In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 408, no 9, p. 2347-2356Article in journal (Refereed)
    Abstract [en]

    Phosphorylation of proteins is important for controlling cellular signaling and cell cycle regulatory events. The process is reversible and phosphoproteins normally constitute a minor part of the global proteome in a cell. Thus, sample preparation techniques tailored for phosphoproteome studies are continuously invented and evaluated. This paper aims at evaluating the performances of the most popular techniques for phospho-enrichments in sub-proteome analysis, such as viral proteomes expressed in human cells during infection. A two-species sample of Adenovirus type 2 infected human cells was used, and in-solution digestion, strong cation exchange (SCX), and electrostatic repulsion hydrophilic interaction chromatography (ERLIC) fractionation, and subsequent enrichment by TiO2, were compared with SDS-PAGE fractionation and in-gel digestion. Evaluation was focused on phosphopeptide detection in the sub-proteome. The results showed that the SCX+TiO2 or ERLIC+TiO2 combinations had the highest enrichment efficiencies, but SDS-PAGE fractionation and in-gel digestion resulted in the highest number of identified proteins and phosphopeptides. Furthermore, the study demonstrates the usefulness of applying as many orthogonal techniques as possible in deep phosphoproteome analysis, since the overlap between approaches was low.

  • 20.
    Källsten, Malin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Recipharm OT Chem AB, Virdings Alle 32b, S-75450 Uppsala, Sweden.
    Pijnappel, Matthijs
    Recipharm OT Chem AB, Virdings Alle 32b, S-75450 Uppsala, Sweden.
    Hartmann, Rafael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preparative Medicinal Chemistry.
    Lehmann, Fredrik
    Oncopeptides AB, Luntmakargatan 46, SE-11137 Stockholm, Sweden.
    Kovac, Lucia
    Recipharm OT Chem AB, Virdings Alle 32b, S-75450 Uppsala, Sweden.
    Lind, Sara
    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.
    Application of triple quadrupole mass spectrometry for the characterization of antibody-drug conjugates2019In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 411, no 12, p. 2569-2576Article in journal (Refereed)
    Abstract [en]

    Antibody-drug conjugates (ADCs) are an inherently heterogeneous class of biotherapeutics, the development of which requires extensive characterization throughout. During the earliest phases of preclinical development, when synthetic routes towards the desired conjugate are being assessed, the main interest lies in the determination of the average drug-to-antibody ratio (DAR) of a given batch as well as information about different conjugation species. There has been a trend in mass spectrometry (MS)-based characterization of ADCs towards the use of high-resolving mass spectrometry for many of these analyses. Considering the high cost for such an instrument, the evaluation of cheaper and more accessible alternatives is highly motivated. We have therefore tested the applicability of a quadrupole mass analyzer for the aforementioned characterizations. Eight ADCs consisting of trastuzumab and varying stoichiometries of Mc-Val-Cit-PABC-monomethyl auristatin E conjugated to native cysteines were synthesized and served as test analytes. The average DAR value and molecular weights (Mw) of all detected chains from the quadrupole mass analyzer showed surprisingly high agreement with results obtained from a time-of-flight (TOF) mass analyzer and hydrophobic interaction chromatography (HIC)-derived values for all investigated ADC batches. Acquired Mw were within 80ppm of TOF-derived values, and DAR was on average within 0.32 DAR units of HIC-derived values. Quadrupole mass spectrometers therefore represent a viable alternative for the characterization of ADC in early-stage development.

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  • 21.
    Lanekoff, Ingela
    et al.
    Physical Sciences Division, Pacific Northwest National Laboratory,Richland, WA 99354, USA .
    Burnum-Johnson, Kristin
    Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99354, USA.
    Thomas, Mathew
    Computational Sciences and Mathematics Division, Pacific Northwest National Laboratory, Richland, WA 99354, USA.
    Cha, Jeeyeon
    Division of Reproductive Sciences, The Perinatal Institute, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA.
    Dey, SudhansuK.
    Division of Reproductive Sciences, The Perinatal Institute, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA.
    Yang, Pengxiang
    Thermo Fisher Scientific, San Jose, CA 95134, USA.
    Prieto Conaway, MariaC.
    Thermo Fisher Scientific, San Jose, CA 95134, USA.
    Laskin, Julia
    Physical Sciences Division, Pacific Northwest National Laboratory,Richland, WA 99354, USA .
    Three-dimensional imaging of lipids and metabolites in tissues by nanospray desorption electrospray ionization mass spectrometry2015In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 407, no 8, p. 2063-2071Article in journal (Refereed)
  • 22. Lanekoff, Ingela
    et al.
    Karlsson, Roger
    Analysis of intact ladderane phospholipids, originating from viable anammox bacteria, using RP-LC-ESI-MS2010In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 397, no 8, p. 3543-3551Article in journal (Refereed)
    Abstract [en]

    Since the discovery of the anaerobic ammonium oxidizing (anammox) bacteria, many attempts have been made in order to identify these environmentally important bacteria in natural environments. Anammox bacteria contain a unique class of lipids, called ladderane lipids and here we present a novel method to detect viable anammox bacteria in sediments and waste water treatment plants based on the use of a ladderane lipid biomarker. Intact ladderane phosphatidylcholine (PC) lipids are analyzed using reversed-phase liquid chromatography-electrospray ionization-mass spectrometry. Following extraction from the complex sediment matrix, reversed-phase LC is used to separate ladderane PC lipids based on their tail group hydrophobicity as well as their ether or ester link to the glycerol backbone in the sn-2 position. We investigate the presence of intact ladderane lipids in natural sediments displaying anammox activity and illustrate the use of a specific intact membrane forming PC lipid as a biomarker for viable anammox bacterial cells. The presented method can be used to elucidate the whereabouts of viable anammox bacteria, subsequently enabling an estimation of anammox activity. This will greatly increase the knowledge of anammox bacteria and their importance in the global nitrogen cycle.

  • 23.
    Leito, Ivo
    et al.
    Univ Tartu, Inst Chem, Ravila 14a, EE-50411 Tartu, Estonia.
    Teearu, Anu
    Univ Tartu, Inst Chem, Ravila 14a, EE-50411 Tartu, Estonia.
    Bobacka, Johan
    Abo Akad Univ, Analyt Chem Lab, Johan Gadolin Proc Chem Ctr, Biskopsgatan 8, SF-20500 Turku, Finland.
    Randon, Jerome
    Univ Claude Bernard Lyon 1, CNRS, UMR 5280, Univ Lyon,Inst Sci Analyt, 5 Rue Doua, F-69100 Villeurbanne, France.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    EACH (Excellence in Analytical Chemistry), an Erasmus Mundus Joint Programme: progress and success2019In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 411, no 23, p. 5913-5921Article in journal (Other academic)
  • 24.
    Lillja, Johan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Lanekoff, Ingela
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Quantitative determination of sn-positional phospholipid isomers in MSn using silver cationization2022In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 414, no 25, p. 7473-7482Article in journal (Refereed)
    Abstract [en]

    Glycerophospholipids are one of the fundamental building blocks for life. The acyl chain connectivity to the glycerol backbone constitutes different sn-positional isomers, which have great diversity and importance for biological function. However, to fully realize their impact on function, analytical techniques that can identify and quantify sn-positional isomers in chemically complex biological samples are needed. Here, we utilize silver ion cationization in combination with tandem mass spectrometry (MSn) to identify sn-positional isomers of phosphatidylcholine (PC) species. In particular, a labile carbocation is generated through a neutral loss (NL) of AgH, the dissociation of which provides diagnostic product ions that correspond to acyl chains at the sn-1 or sn-2 position. The method is comparable to currently available methods, has a sensitivity in the nM-mu M range, and is compatible with quantitative imaging using mass spectrometry in MS4. The results reveal a large difference in isomer concentrations and the ion images show that the sn-positional isomers PC 18:1_18:0 are homogeneously distributed, whereas PC 18:1_16:0 and PC 20:1_16:0 show distinct localizations to sub-hippocampal structures.

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  • 25.
    Lim, Hwanmi
    et al.
    Stockholm Univ, Dept Environm Sci & Analyt Chem ACES, S-10691 Stockholm, Sweden..
    Ahmed, Trifa M.
    Stockholm Univ, Dept Environm Sci & Analyt Chem ACES, S-10691 Stockholm, Sweden.;Livsmedelsverket, Box 622, S-75126 Uppsala, Sweden..
    Bergvall, Christoffer
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology. Stockholm Univ, Dept Environm Sci & Analyt Chem ACES, S-10691 Stockholm, Sweden..
    Westerholm, Roger
    Stockholm Univ, Dept Environm Sci & Analyt Chem ACES, S-10691 Stockholm, Sweden..
    Automated clean-up, separation and detection of polycyclic aromatic hydrocarbons in particulate matter extracts using a 2D-LC/2D-GC system: a method translation from two FIDs to two MS detectors2017In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 409, no 24, p. 5619-5629Article in journal (Refereed)
    Abstract [en]

    An online two-dimensional (2D) liquid chromatography/2D gas chromatography system with two mass-selective detectors has been developed on the basis of a previous system with two flame ionization detectors. The method translation involved the change of carrier gas from hydrogen to helium, column dimension and detectors. The 2D system with two mass-selective detectors was validated with use of polycyclic aromatic hydrocarbon (PAH) standards and two standard reference materials from air and diesel exhaust. Furthermore, the system was applied to a real sample, wood smoke particulates. The PAH values determined correlated well with the previous data and those from the National Institute of Standards and Technology. The system enhanced the benefits of the previous system, which were limited by the low detectability and lack of mass selectivity. This study shows an automated 2D system that is valid for PAH analysis of complex environmental samples directly from crude extracts.

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  • 26. Lindholm-Sethson, Britta
    et al.
    Nyström, Josefina
    Malmsten, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Ringstad, Lovisa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Nelson, Andrew
    Geladi, Paul
    Electrochemical impedance spectroscopy in label-free biosensor applications: multivariate data analysis for an objective interpretation2010In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 398, no 6, p. 2341-2349Article, review/survey (Refereed)
    Abstract [en]

    Electrochemical impedance spectroscopy plays an important role in biosensor science thanks to the possibility of finding specific information from processes with different kinetics at a chosen electrode potential in one experiment. In this paper we briefly discuss label-free impedimetric biosensors described in the literature. A novel method for neutral interpretation of impedance data is presented that includes complex number chemometrics. Three examples are given based on impedance measurements on synthetic biomembranes, in this case a lipid monolayer deposited on a mercury electrode. The interaction of various compounds with the monomolecular lipid layer is illustrated with the following: (1) different concentrations of magainin (Geladi et al. in Proc. Int. Fed. Med. Biomed. Eng. 9:219-220, 2005); (2) different derivatives of gramicidin A (Lindholm-Sethson et al. in Langmuir 24:5029-5032, 2007), and (3) an antimicrobial peptide (Ringstad et al. in Langmuir 24:208-216, 2008).

  • 27. Liu, Jiayin
    et al.
    Sandahl, Margareta
    Sjöberg, Per J. R.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Turner, Charlotta
    Pressurised hot water extraction in continuous flow mode for thermolabile compounds: extraction of polyphenols in red onions2014In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 406, no 2, p. 441-445Article in journal (Refereed)
    Abstract [en]

    Extraction and analysis of labile compounds in complex sample matrices, such as plants, is often a big analytical challenge. In this work, the use of a "green and clean" pressurised hot water extraction (PHWE) approach performed in continuous flow mode is explored. Experimental data for extraction and degradation kinetics of selected compounds were utilised to develop a continuous flow extraction (CFE) method targeting thermolabile polyphenols in red onions, with detection by high-performance liquid chromatography (HPLC)-diode array detection (DAD)-mass spectrometry (MS). Water containing ethanol and formic acid was used as extraction solvent. Method performance was focused on extraction yield with minimal analyte degradation. By adjusting the flow rate of the extraction solvent, degradation effects were minimised, and complete extraction could be achieved within 60 min. The CFE extraction yields of the polyphenols investigated were 80-90 % of the theoretically calculated quantitative yields and were significantly higher than the yields obtained by conventional methanol extraction and static batch extraction (70-79 and 58-67% of the theoretical yields, respectively). The precision of the developed method was lower than 8 % expressed as relative standard deviation.

  • 28.
    Lundkvist, Stefan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC. Thomas Jefferson Univ, Sidney Kimmel Med Coll, Jefferson Inst Mol Med, Dept Dermatol & Cutaneous Biol, 233 S 10th St, Philadelphia, PA 19107 USA.;Thomas Jefferson Univ, Sidney Kimmel Med Coll, PXE Int Ctr Excellence Res & Clin Care, 233 S 10th St, Philadelphia, PA 19107 USA..
    Niaziorimi, Fatemeh
    Thomas Jefferson Univ, Sidney Kimmel Med Coll, Jefferson Inst Mol Med, Dept Dermatol & Cutaneous Biol, 233 S 10th St, Philadelphia, PA 19107 USA.;Thomas Jefferson Univ, Sidney Kimmel Med Coll, PXE Int Ctr Excellence Res & Clin Care, 233 S 10th St, Philadelphia, PA 19107 USA..
    Szeri, Flora
    Thomas Jefferson Univ, Sidney Kimmel Med Coll, Jefferson Inst Mol Med, Dept Dermatol & Cutaneous Biol, 233 S 10th St, Philadelphia, PA 19107 USA.;Thomas Jefferson Univ, Sidney Kimmel Med Coll, PXE Int Ctr Excellence Res & Clin Care, 233 S 10th St, Philadelphia, PA 19107 USA.;Inst Enzymol, Res Ctr Nat Sci, Budapest, Hungary.;Semmelweis Univ, Dept Biochem, Budapest, Hungary..
    Caffet, Matthew
    PXE Int, Damascus, MD USA..
    Terry, Sharon F.
    PXE Int, Damascus, MD USA..
    Johansson, Gunnar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Jansen, Robert S.
    Radboud Univ Nijmegen, Dept Microbiol, Nijmegen, Netherlands..
    van de Wetering, Koen
    Thomas Jefferson Univ, Sidney Kimmel Med Coll, Jefferson Inst Mol Med, Dept Dermatol & Cutaneous Biol, 233 S 10th St, Philadelphia, PA 19107 USA.;Thomas Jefferson Univ, Sidney Kimmel Med Coll, PXE Int Ctr Excellence Res & Clin Care, 233 S 10th St, Philadelphia, PA 19107 USA..
    A new enzymatic assay to quantify inorganic pyrophosphate in plasma2023In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 415, no 3, p. 481-492Article in journal (Refereed)
    Abstract [en]

    Inorganic pyrophosphate (PPi) is a crucial extracellular mineralization regulator. Low plasma PPi concentrations underlie the soft tissue calcification present in several rare hereditary mineralization disorders as well as in more common conditions like chronic kidney disease and diabetes. Even though deregulated plasma PPi homeostasis is known to be linked to multiple human diseases, there is currently no reliable assay for its quantification. We here describe a PPi assay that employs the enzyme ATP sulfurylase to convert PPi into ATP. Generated ATP is subsequently quantified by firefly luciferase-based bioluminescence. An internal ATP standard was used to correct for sample-specific interference by matrix compounds on firefly luciferase activity. The assay was validated and shows excellent precision (< 3.5%) and accuracy (93-106%) of PPi spiked into human plasma samples. We found that of several anticoagulants tested only EDTA effectively blocked conversion of ATP into PPi in plasma after blood collection. Moreover, filtration over a 300,000-Da molecular weight cut-off membrane reduced variability of plasma PPi and removed ATP present in a membrane-enclosed compartment, possibly platelets. Applied to plasma samples of wild-type and Abcc6(-/-) rats, an animal model with established low circulating levels of PPi, the new assay showed lower variability than the assay that was previously in routine use in our laboratory. In conclusion, we here report a new and robust assay to determine PPi concentrations in plasma, which outperforms currently available assays because of its high sensitivity, precision, and accuracy.

  • 29.
    Lönnberg, Maria
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Bondesson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Cormant, Florence
    L.C.H., Laboratoire des Courses Hippiques, France.
    Garcia, Patrice
    L.C.H., Laboratoire des Courses Hippiques, France.
    Bonnaire, Yves
    L.C.H., Laboratoire des Courses Hippiques, France.
    Carlsson, Jan
    MAIIA Diagnostics, Uppsala, Sweden.
    Popot, Marie-Agnes
    L.C.H., Laboratoire des Courses Hippiques, France.
    Rollborn, Niclas
    MAIIA Diagnostics, Uppsala, Sweden.
    Råsbo, Kristina
    Bailly-Chouriberry, Ludovic
    L.C.H., Laboratoire des Courses Hippiques, France.
    Detection of recombinant human EPO administered to horses using MAIIA lateral flow isoform test2012In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 403, no 6, p. 1619-1628Article in journal (Refereed)
    Abstract [en]

    Doping of horses with recombinant human erythropoietin (rHuEPO) to illegally enhance their endurance capacity in horseracing has been reported during the last years. This leads to increased blood viscosity which can result in sudden death and is of concern for the horse welfare. Additionally, the horse can start production of rHuEPO antibodies, which cross-reacts with endogenous equine EPO and can lead to severe anaemia and even death. In this study, a novel micro-chromatographic method, EPO WGA MAIIA, has been tested for the capability in plasma and urine samples to detect administration of erythropoiesis-stimulating agents, like the rHuEPO glycoprotein varieties Eprex and Aranesp, to horses. After administration of 40 IU Eprex kg −1 day −1 to seven horses during 6 days, the presence of Eprex in horse plasma was detected up to 2–5 days after last injection. In urine samples collected from two horses, Eprex was detected up to 3 days. A single injection of Aranesp (0.39 μg/kg) was detected up to 9 days in plasma and up to 8 days, the last day of testing, in the urine sample. The LC-FAIMS-MS/MS system, with 1 day reporting time, confirmed the presence of Eprex up to 1 day after last injection for six out of seven horses and the presence of Aranesp up to 5 days after last injection in plasma samples. The MAIIA system showed to be a promising tool with high sensitivity and extremely short reporting time (1 h).

  • 30.
    Lönnberg, Maria
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Lundby, Carsten
    Center for Integrative Human Physiology (ZIHP), Institute of Physiology, University of Zurich, Switzerland.
    Detection of EPO injections using a rapid lateral flow isoform test2013In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 405, no 30, p. 9685-9691Article in journal (Refereed)
    Abstract [en]

    Misuse of recombinant human erythropoietin (rhEPO) is a major concern in competitive sports, and the implementation of tests allowing for higher detection rates than what current tests are capable of is required. In this study, a novel lateral flow EPO isoform test kit, EPO WGA MAIIA, is evaluated on the basis of plasma and urine samples obtained from eight healthy males in connection with a 28-day rhEPO injection period. rhEPO was injected every other day during the first 14 days of the study, and the method proved to be 100 % effective in detecting rhEPO in the concomitantly obtained samples. Seven days after the last injection, three positive (>99.99 % confidence limit (CL)) subjects were found. When using 99 % CL as the cut-off limit, six of the eight subjects (75 %) were found to be suspected of doping. Samples obtained 14 and 21 days after the last injection showed no detectable trace of rhEPO. A previous study using indirect methods to determine EPO doping on the same samples indicated only that two of the subjects had suspicious values 7-21 days after the last injection. We propose implementing the easy to-use EPO WGA MAIIA test as an initial screening procedure in anti-doping work to (1) increase the detection rate of potential rhEPO doping athletes and (2) allow for a 10- to 20-fold higher analytical rate than what is possible today.

  • 31. Maddalo, Gianluca
    et al.
    Shariatgorji, Mohammadreza
    Adams, Christopher M.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Fung, Eva
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Nilsson, Ulrika
    Zubarev, Roman A.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Sedzik, Jan
    Ilag, Leopold L.
    Porcine P2 myelin protein primary structure and bound fatty acids determined by mass spectrometry2010In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 397, no 5, p. 1903-1910Article in journal (Refereed)
    Abstract [en]

    Complementary collision-induced/electron capture dissociation Fourier-transform ion cyclotron resonance mass spectrometry was used to fully sequence the protein P2 myelin basic protein. It is an antigenic fatty-acid-binding protein that can induce experimental autoimmune neuritis: an animal model of Guillain-Barre syndrome, a disorder similar in etiology to multiple sclerosis. Neither the primary structure of the porcine variant, nor the fatty acids bound by the protein have been well established to date. A 1.8-angstrom crystal structure shows but a bound ligand could not be unequivocally identified. A protocol for ligand extraction from protein crystals has been developed with subsequent gas chromatography MS analysis allowing determination that oleic, stearic, and palmitic fatty acids are associated with the protein. The results provide unique and general evidence of the utility of mass spectrometry for characterizing proteins from natural sources and generating biochemical information that may facilitate attempts to elucidate the causes for disorders such as demyelination.

  • 32.
    Mavroudakis, Leonidas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Stevens, Susan
    Duncan, Kyle D.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Stenzel-Poore, Mary
    Laskin, Julia
    Lanekoff, Ingela
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    CpG preconditioning reduces accumulation of lysophosphatidylcholine in ischemic brain tissue after middle cerebral artery occlusion2021In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 413, p. 2735-2745Article in journal (Refereed)
    Abstract [en]

    Ischemic stroke is one of the major causes of death and permanent disability in the world. However, the molecular mechanisms surrounding tissue damage are complex and further studies are needed to gain insights necessary for development of treatment. Prophylactic treatment by administration of cytosine-guanine (CpG) oligodeoxynucleotides has been shown to provide neuroprotection against anticipated ischemic injury. CpG binds to Toll-like receptor 9 (TLR9) causing initialization of an inflammatory response that limits visible ischemic damages upon subsequent stroke. Here, we use nanospray desorption electrospray ionization (nano-DESI) mass spectrometry imaging (MSI) to characterize molecular effects of CpG preconditioning prior to middle cerebral artery occlusion (MCAO) and reperfusion. By doping the nano-DESI solvent with appropriate internal standards, we can study and compare distributions of phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) in the ischemic hemisphere of the brain despite the large changes in alkali metal abundances. Our results show that CpG preconditioning not only reduces the infarct size but it also decreases the degradation of PC and accumulation of LPC species, which indicates reduced cell membrane breakdown and overall ischemic damage. Our findings show that molecular mechanisms of PC degradation are intact despite CpG preconditioning but that these are limited due to the initialized inflammatory response.

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  • 33. McDonnell, Liam A.
    et al.
    Roempp, Andreas
    Balluff, Benjamin
    Heeren, Ron M. A.
    Albar, Juan Pablo
    Andrén, Per E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Corthals, Garry L.
    Walch, Axel
    Stoeckli, Markus
    Discussion point: reporting guidelines for mass spectrometry imaging2015In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 407, no 8, p. 2035-2045Article in journal (Refereed)
  • 34.
    Meiby, Elinor
    et al.
    Department of Chemistry and Biomedical Sciences, Linnaeus University, Kalmar.
    Morin Zetterberg, Malin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Ohlson, Sten
    Department of Chemistry and Biomedical Sciences, Linnaeus University, Kalmar Sweden AND School of Biological Sciences, Nanyang Technological University, Singapore.
    Agmo Hernández, Víctor
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Edwards, Katarina
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Immobilized lipodisks as model membranes in high-throughput HPLC-MS analysis2013In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 405, no 14, p. 4859-4869Article in journal (Refereed)
    Abstract [en]

    Lipodisks, also referred to as polyethylene glycol (PEG)-stabilized bilayer disks, have previously been demonstrated to hold great potential as model membranes in drug partition studies. In this study, an HPLC-MS system with stably immobilized lipodisks is presented. Functionalized lipodisks were immobilized on two different HPLC support materials either covalently by reductive amination or by streptavidin-biotin binding. An analytical HPLC column with immobilized lipodisks was evaluated by analysis of mixtures containing 15 different drug compounds. The efficiency, reproducibility, and stability of the system were found to be excellent. In situ incorporation of cyclooxygenase-1 (COX-1) in immobilized lipodisks on a column was also achieved. Specific binding of COX-1 to the immobilized lipodisks was validated by interaction studies with QCM-D. These results, taken together, open up the possibility of studying ligand interactions with membrane proteins by weak affinity chromatography.

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  • 35.
    Mohabbati, S
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Hjertén, S
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Westerlund, D
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Studies on the analytical performance of a non-covalent coating with N,N-didodecyl-N,N-dimethylammonium bromide for separation of basic proteins by capillary electrophoresis in acidic buffers in 25- and 50-microm capillaries2008In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 390, no 2, p. 667-678Article in journal (Refereed)
    Abstract [en]

    Capillaries (25-and 50-mu m inner diameter) coated with a double-alkyl-chain cationic surfactant N,N-didodecyl-N,N-dimethylammonium bromide (DDAB) were used for the separation of four basic standard proteins in buffers of pH 4 at various ionic strengths. The choice of buffer is critical for the analytical performance. Ammonium ions must be avoided in the buffer used in the non-covalent coating procedure owing to competition with the surfactant. Phosphate buffer gave a better separation performance than some volatile buffers; the reason seems to be a complex formation between the proteins and dihydrogenphosphate ions, which decreases tendencies for adsorption to the capillary surface. The DDAB coating was easy to produce and stable enough to permit, without recoating, consecutive separations of the proteins for up to 100 min with good precision in migration times and peak areas. A strong electroosmotic flow gives rapid separations, which is of special importance when commercial instruments are used, since the choice of the length of the capillary is restricted.

  • 36.
    Musunuri, Sravani
    et al.
    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.
    Richard, Bernhard Clemens
    Ingelsson, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Shevchenko, Ganna
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Micellar extraction possesses a new advantage for the analysis of Alzheimer's disease brain proteome2015In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 407, no 4, p. 1041-1057Article in journal (Refereed)
    Abstract [en]

    Integral membrane proteins (MPs), such as transporters, receptors, and ion channels, are of great interest because of their participation in various vital cellular functions including cell-cell interactions, ion transport, and signal transduction. However, studies of MPs are complicated because of their hydrophobic nature, heterogeneity, and low abundance. Cloud-point extraction (CPE) with the non-ionic surfactant Triton X-114 was performed to simultaneously extract and phase separate hydrophobic and hydrophilic proteins from Alzheimer's disease (AD) and unaffected control brain tissue. Quantitative proteomics analysis of temporal neocortex samples of AD patients and controls was performed using a shotgun approach based on stable isotope dimethyl labeling (DML) quantification technique followed by nanoLC-MS/MS analysis. A total of 1096 unique proteins were identified and quantified, with 40.3 % (211/524) predicted as integral MPs with at least one transmembrane domain (TMD) found in the detergent phase, and 10 % (80/798) in the detergent-depleted phase. Among these, 62 proteins were shown to be significantly altered (p-value < 0.05), in AD versus control samples. In the detergent fraction, we found 10 hydrophobic transmembrane proteins containing up to 14 putative TMDs that were significantly up- or down-regulated in AD compared with control brains. Changes in four of these proteins, alpha-enolase (ENOA), lysosome-associated membrane glycoprotein 1 (LAMP1), 14-3-3 protein gamma (1433G), and sarcoplasmic/endoplasmic reticulum calcium ATPase2 (AT2A2) were validated by immunoblotting. Our results emphasize that separating hydrophobic MPs in CPE contributes to an increased understanding of the underlying molecular mechanisms in AD. Such knowledge can become useful for the development of novel disease biomarkers.

  • 37.
    Nicholls, Ian A.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Andersson, Håkan S.
    Golker, Kerstin
    Henschel, Henning
    Karlsson, Björn C. G.
    Olsson, Gustaf D.
    Rosengren, Annika M.
    Shoravi, Siamak
    Suriyanarayanan, Subramanian
    Wiklander, Jesper G.
    Wikman, Susanne
    Rational design of biomimetic molecularly imprinted materials: theoretical and computational strategies for guiding nanoscale structured polymer development2011In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 400, no 6, p. 1771-1786Article, review/survey (Refereed)
    Abstract [en]

    In principle, molecularly imprinted polymer science and technology provides a means for ready access to nano-structured polymeric materials of predetermined selectivity. The versatility of the technique has brought it to the attention of many working with the development of nanomaterials with biological or biomimetic properties for use as therapeutics or in medical devices. Nonetheless, the further evolution of the field necessitates the development of robust predictive tools capable of handling the complexity of molecular imprinting systems. The rapid growth in computer power and software over the past decade has opened new possibilities for simulating aspects of the complex molecular imprinting process. We present here a survey of the current status of the use of in silico-based approaches to aspects of molecular imprinting. Finally, we highlight areas where ongoing and future efforts should yield information critical to our understanding of the underlying mechanisms sufficient to permit the rational design of molecularly imprinted polymers.

  • 38.
    Nilsson, Kelly Dimovska
    et al.
    Univ Gothenburg, Dept Chem & Mol Biol, S-40530 Gothenburg, Sweden..
    Karagianni, Anthi
    Univ Gothenburg, Dept Chem & Mol Biol, S-40530 Gothenburg, Sweden..
    Kaya, Ibrahim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Univ Gothenburg, Dept Chem & Mol Biol, S-40530 Gothenburg, Sweden.;Univ Gothenburg, Dept Psychiat & Neurochem, Sahlgrenska Acad, S-41345 Mölndal, Sweden..
    Henricsson, Marcus
    Univ Gothenburg, Inst Med, Dept Mol & Clin Med, Wallenberg Lab, S-41345 Gothenburg, Sweden..
    Fletcher, John S.
    Univ Gothenburg, Dept Chem & Mol Biol, S-40530 Gothenburg, Sweden..
    (CO2)(n)(+), (H2O)(n)(+), and (H2O)(n)(+) (CO2) gas cluster ion beam secondary ion mass spectrometry: analysis of lipid extracts, cells, and Alzheimer's model mouse brain tissue2021In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 413, no 16, p. 4181-4194Article in journal (Refereed)
    Abstract [en]

    This work assesses the potential of new water cluster-based ion beams for improving the capabilities of secondary ion mass spectrometry (SIMS) for in situ lipidomics. The effect of water clusters was compared to carbon dioxide clusters, along with the effect of using pure water clusters compared to mixed water and carbon dioxide clusters. A signal increase was found when using pure water clusters. However, when analyzing cells, a more substantial signal increase was found in positive ion mode when the water clusters also contained carbon dioxide, suggesting that additional reactions are in play. The effects of using a water primary ion beam on a more complex sample were investigated by analyzing brain tissue from an Alzheimer's disease transgenic mouse model. The results indicate that the ToF-SIMS results are approaching those from MALDI as ToF-SIMS was able to image lyso-phosphocholine (LPC) lipids, a lipid class that for a long time has eluded detection during SIMS analyses. Gangliosides, sulfatides, and cholesterol were also imaged.

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  • 39.
    Pasquini, Chiara
    et al.
    Free Univ Berlin, Dept Phys, Arnimallee 14, D-14195 Berlin, Germany..
    Liu, Si
    Free Univ Berlin, Dept Phys, Arnimallee 14, D-14195 Berlin, Germany..
    Chernev, Petko
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics. Free Univ Berlin, Dept Phys, Arnimallee 14, D-14195 Berlin, Germany..
    Gonzalez-Flores, Diego
    Free Univ Berlin, Dept Phys, Arnimallee 14, D-14195 Berlin, Germany.;Univ Costa Rica, Ctr Electroquim & Energia Quim CELEQ, San Jose 115012060, Costa Rica.;Univ Costa Rica, Escuela Quim, San Jose 115012060, Costa Rica..
    Mohammadi, Mohammad Reza
    Free Univ Berlin, Dept Phys, Arnimallee 14, D-14195 Berlin, Germany.;Univ Sistan & Baluchestan, Dept Phys, Zahedan 9816745845, Iran..
    Kubella, Paul
    Free Univ Berlin, Dept Phys, Arnimallee 14, D-14195 Berlin, Germany..
    Jiang, Shan
    Free Univ Berlin, Dept Phys, Arnimallee 14, D-14195 Berlin, Germany..
    Loos, Stefan
    Free Univ Berlin, Dept Phys, Arnimallee 14, D-14195 Berlin, Germany.;Fraunhofer Inst Mfg Technol & Adv Mat IFAM, Winterbergstr 28, D-01277 Dresden, Germany..
    Klingan, Katharina
    Free Univ Berlin, Dept Phys, Arnimallee 14, D-14195 Berlin, Germany..
    Sikolenko, Vadim
    Free Univ Berlin, Dept Phys, Arnimallee 14, D-14195 Berlin, Germany.;Karlsruhe KIT, Karlsruhe Inst Technol, Adenauerring 20, D-76131 Karlsruhe, Germany..
    Mebs, Stefan
    Free Univ Berlin, Dept Phys, Arnimallee 14, D-14195 Berlin, Germany..
    Haumann, Michael
    Free Univ Berlin, Dept Phys, Arnimallee 14, D-14195 Berlin, Germany..
    Beyer, Paul
    Free Univ Berlin, Dept Phys, Arnimallee 14, D-14195 Berlin, Germany..
    D'Amario, Luca
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics. Free Univ Berlin, Dept Phys, Arnimallee 14, D-14195 Berlin, Germany..
    Smith, Rodney D. L.
    Free Univ Berlin, Dept Phys, Arnimallee 14, D-14195 Berlin, Germany.;Univ Waterloo, Dept Chem, 200 Univ Ave W, Waterloo, ON N2L 3G1, Canada..
    Zaharieva, Ivelina
    Free Univ Berlin, Dept Phys, Arnimallee 14, D-14195 Berlin, Germany..
    Dau, Holger
    Free Univ Berlin, Dept Phys, Arnimallee 14, D-14195 Berlin, Germany..
    Operando tracking of oxidation-state changes by coupling electrochemistry with time-resolved X-ray absorption spectroscopy demonstrated for water oxidation by a cobalt-based catalyst film2021In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 413, no 21, p. 5395-5408Article in journal (Refereed)
    Abstract [en]

    Transition metal oxides are promising electrocatalysts for water oxidation, i.e., the oxygen evolution reaction (OER), which is critical in electrochemical production of non-fossil fuels. The involvement of oxidation state changes of the metal in OER electrocatalysis is increasingly recognized in the literature. Tracing these oxidation states under operation conditions could provide relevant information for performance optimization and development of durable catalysts, but further methodical developments are needed. Here, we propose a strategy to use single-energy X-ray absorption spectroscopy for monitoring metal oxidation-state changes during OER operation with millisecond time resolution. The procedure to obtain time-resolved oxidation state values, using two calibration curves, is explained in detail. We demonstrate the significance of this approach as well as possible sources of data misinterpretation. We conclude that the combination of X-ray absorption spectroscopy with electrochemical techniques allows us to investigate the kinetics of redox transitions and to distinguish the catalytic current from the redox current. Tracking of the oxidation state changes of Co ions in electrodeposited oxide films during cyclic voltammetry in neutral pH electrolyte serves as a proof of principle.

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  • 40.
    Samgina, Tatiana Yu
    et al.
    Moscow MV Lomonosov State Univ, Dept Chem, Leninskie Gori 1-3, Moscow 119991, Russia..
    Artemenko, Konstantin A.
    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.
    Trebse, Polonca
    Univ Ljubljana, Fac Hlth Sci, Zdravstvena Pot 5, Ljubljana 1000, Slovenia..
    Torkar, Gregor
    Univ Ljubljana, Fac Educ, Kardeljeva Ploscad 16, Ljubljana 1000, Slovenia..
    Tolpina, Miriam D.
    Moscow MV Lomonosov State Univ, Dept Chem, Leninskie Gori 1-3, Moscow 119991, Russia..
    Lebedev, Albert T.
    Moscow MV Lomonosov State Univ, Dept Chem, Leninskie Gori 1-3, Moscow 119991, Russia..
    Differentiation of frogs from two populations belonging to the Pelophylax esculentus complex by LC-MS/MS comparison of their skin peptidomes2017In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 409, no 7, p. 1951-1961Article in journal (Refereed)
    Abstract [en]

    LC-MS/MS was applied to establish the composition of the skin peptidome of a Slovenian green frog belonging to the Pelophylax esculentus complex. As this was similar to the peptidome of the Moscow population of Pelophylax ridibundus, it allowed us to identify the Slovenian frog from the Pelophylax esculentus complex as Pelophylax ridibundus. The sequences of six new peptides from the brevinin 2 family are reported for the first time on the basis of manual interpretation of their tandem mass spectra. The structural similarity of the brevinin 2 peptides from the Moscow and Slovenian populations of Pelophylax ridibundus enables peptides from this family to be utilized as biomarkers for Pelophylax ridibundus inter- and intraspecies differentiation, and the proposed approach can be used as an analytical tool for differentiating the corresponding species and populations. The potential biological activities of the novel peptides were estimated by 2D mass mapping. The results allowed us to classify all of the available peptides belonging to the brevinin 2 family.

  • 41.
    Samgina, Tatiana Yu
    et al.
    Moscow MV Lomonosov State Univ, Dept Organ Chem, Moscow 119991, Russia..
    Tolpina, Miriam I.
    Moscow MV Lomonosov State Univ, Dept Organ Chem, Moscow 119991, Russia..
    Hakalehto, Elias
    Univ Eastern Finland, Dept Environm Sci, POB 1627, Kuopio 70211, Finland..
    Artemenko, Konstantin A.
    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.
    Lebedev, Albert T.
    Moscow MV Lomonosov State Univ, Dept Organ Chem, Moscow 119991, Russia..
    Proteolytic degradation and deactivation of amphibian skin peptides obtained by electrical stimulation of their dorsal glands2016In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 408, no 14, p. 3761-3768Article in journal (Refereed)
    Abstract [en]

    Amphibians are among the oldest creatures on our planet. Their only defensive weapon efficient against microorganisms and predators involves their skin secretion. The wide range of biological activities of the peptides in the skin secretion of amphibians makes these compounds rather interesting for generation of prospective pharmaceuticals. The first step in studying these molecules requires their structures to be established. Mass spectrometry is the most powerful tool for this purpose. The sampling and sample preparation stages preceding mass spectrometry experiments appear to be rather crucial. The results obtained here demonstrate that these preparation procedures might lead to partial or complete loss of the bioactive peptides in the secretion. Five minutes in water was enough to completely destroy all of the bioactive peptides in the skin secretion of the marsh frog (Rana ridibunda); even immediate addition of methanol to the water solution of the peptides did not prevent partial destruction. Concerted effort should be directed towards development of the most efficient procedure to keep the secreted peptides intact.

  • 42.
    Shariatgorji, Mohammadreza
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Astorga-Wells, Juan
    Ilag, Leopold L.
    Trends in the bioanalytical applications of microfluidic electrocapture2011In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 399, no 1, p. 191-195Article in journal (Other academic)
    Abstract [en]
    Downscaled analytical tools for sample preparation have offered benefits such as higher throughput, easier automation and lower sample/reagent consumption. Microfluidic electrocapture, which is a newly developed sample preparation/manipulation system, uses an electric field to trap and separate charged species without using any solid sorbent. The feasibility of using microfluidic electrocapture is reported for separation, clean-up, concentration, microreactions and complexation studies of proteins, peptides and other biologically important biomolecules. The instrumentation and applications of microfluidic electrocapture are reviewed and an overview is provided of future perspectives offered by the current and envisaged platforms.
  • 43. Singer, David
    et al.
    Soininen, Hilkka
    Alafuzoff, Irina
    Department of Neuroscience and Neurology, University of Kuopio Finland .
    Hoffmann, Ralf
    Immuno-PCR-based quantification of multiple phosphorylated tau-epitopes linked to Alzheimer's disease2009In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 395, no 7, p. 2263-2267Article in journal (Refereed)
    Abstract [en]

    Several lines of evidence suggest that quantification of phosphorylated sites in the tau-protein (phospho-tau) might be favorable for early and specific Alzheimer's disease diagnosis. The typical setup to quantify phosphorylated tau-epitopes relies on a sandwich ELISA with a capture antibody (Ab) recognizing tau independent of its phosphorylation status and a detector Ab binding specifically to a certain phosphorylation site. Besides Ab specificities, major challenges arise from the very low tau-concentrations in cerebrospinal fluid (CSF) ranging from 100 to 2,000 pg/ml. Based on the phosphorylation degree of a given position, which can be below 10%, the corresponding phospho-tau-level might be much lower, especially for multiphosphorylated epitopes studied here. Thus, a novel, highly sensitive, and generally applicable immunoassay is described to quantify tau-versions, which are phosphorylated at pThr212/pSer214/pThr231/pSer235, down to tau-concentrations of 2 pg/ml in CSF.

  • 44.
    Tevell Åberg, Annica
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry. Natl Vet Inst SVA, Dept Chem Environm & Feed Hyg, S-75189 Uppsala, Sweden..
    Karlsson, Ida
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry. Natl Vet Inst SVA, Dept Chem Environm & Feed Hyg, S-75189 Uppsala, Sweden..
    Hedeland, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry. Natl Vet Inst SVA, Dept Chem Environm & Feed Hyg, S-75189 Uppsala, Sweden..
    Modification and validation of the Endopep-mass spectrometry method for botulinum neurotoxin detection in liver samples with application to samples collected during animal botulism outbreaks2021In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 413, no 2, p. 345-354Article in journal (Refereed)
    Abstract [en]

    Botulinum neurotoxins (BoNTs) are the most potent toxins known and they cause the paralytic disease botulism in humans and animals. In order to diagnose botulism, active BoNT must be detected in biological material. Endopep-MS is a sensitive and selective method for serum samples, based on antibody capture, enzymatic cleavage of target peptides, and detection of cleavage products using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). In many cases of animal botulism, serum samples are not available or they do not contain detectable amounts of BoNT and liver sampling is an alternative for postmortem examinations. However, the Endopep-MS method is impaired by the inherent protease activity of liver samples. In the presented study, the Endopep-MS method has been successfully modified and validated for analysis of cattle, horse, and avian liver samples, introducing a combination of a salt washing step and a protease inhibitor cocktail. These modifications resulted in a substantial decrease in interfering signals and increase in BoNT-specific signals. This led to a substantial improvement in sensitivity for especially BoNT-C and C/D which are among the most prominent serotypes for animal botulism. Botulism was diagnosed with the new method in liver samples from dead cattle and birds from outbreaks in Sweden.

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  • 45.
    Ubhayasekera, Sarojini J.K.A.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Staaf, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health, Pediatrics.
    Forslund, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health, Pediatrics.
    Bergsten, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Free fatty acid determination in plasma by GC-MS after conversion to Weinreb amides2013In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 405, no 6, p. 1929-1935Article in journal (Refereed)
    Abstract [en]

    Circulating free fatty acids (FFAs) play important physiological roles as contributing components in cellular structure as well as energy utilization. Elevated levels of circulating FFAs are associated with metabolic aberrations in humans. FFAs differ in chain length and saturation and may be altered in metabolically dysregulated conditions, such as type 2 diabetes mellitus. Potentially, alterations in circulating levels of specific FFAs could also be important in terms of prognostic value. Various methods have been used to analyze FFAs. In this study, a straightforward and accurate method for the determination of FFAs in plasma has been established and evaluated, through conversion of plasma FFAs into acid fluorides followed by conversion to Weinreb amides (dimethylamide). The method is mild, efficient, selective, and quantitative for FFAs, when analyzed with capillary gas chromatography tandem mass spectrometry. Standard curves were linear over the range of 1,000–20,000 ng/mL with a correlation coefficient (r2) of 0.998, and coefficient of variation of triplicate analysis was <10 %. The gas chromatography–mass spectrometry (GC-MS) technique was reproducible and repeatable, and recoveries were above 90 %. From the generated MS spectra, five specific FFAs were identified. An explicit interest was the quantification of palmitate (C16:0) and palmitoleate (C16:1), which have been connected with detrimental and positive effects on the insulin-producing beta cells, respectively. The results demonstrate the suitability of Weinreb amides for efficient and rapid isolation of FFAs in plasma, prior to quantitative GC-MS analysis. We suggest that the method can be used as a routine standardized way of quantifying FFAs.

  • 46.
    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.

  • 47. Wiberg, Henning
    et al.
    Ek, Patrik
    Ekholm Pettersson, Frida
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Emmer, Åsa
    Roeraade, Johan
    Separation and characterization of aggregated species of amyloid-beta peptides2010In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 397, no 6, p. 2357-2366Article in journal (Refereed)
    Abstract [en]

    We have investigated the use of isoelectric focusing and immunodetection for the separation of low molecular weight species of amyloid-beta (A beta) peptides from their aggregates. From solutions of A beta(1-40) or A beta(1-42) monomeric peptides, low molecular weight material appeared at a pI value of ca. 5, while the presence of aggregates was detected as bands, observed at a pI of 6-6.5. The formation of A beta aggregates (protofibrils) was verified by a sandwich ELISA, employing the protofibril conformation-selective antibody mAb158. In order to study the aggregation behavior when using a mixture of the monomers, we utilized the IEF separation combined with Western blot using two polyclonal antisera, selective for A beta(1-40) and A beta(1-42), respectively. We conclude that both monomers were incorporated in the aggregates. In a further study of the mixed aggregates, we used the protofibril conformation-selective antibody mAb158 for immunoprecipitation, followed by nanoelectrospray mass spectrometry (IP-MS). This showed that the A beta(1-42) peptide is incorporated in the aggregate in a significantly larger proportion than its relative presence in the original monomer composition. IP-MS with mAb158 was also performed, and compared to IP-MS with the A beta-selective antibody mAb1C3, where a monomeric A beta(1-16) peptide was added to the protofibril preparation. A beta(1-16) is known for its poor aggregation propensity, and acted therefore as a selectivity marker. The results obtained confirmed the protofibril conformation selectivity of mAb158.

  • 48. Wiklander, Jesper
    et al.
    Karlsson, Björn C. G.
    Aastrup, Teodor
    Nicholls, Ian A.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Towards a synthetic avidin mimic2011In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 400, no 5, p. 1397-1404Article in journal (Refereed)
    Abstract [en]

    A series of streptavidin-mimicking molecularly imprinted polymers has been developed and evaluated for their biotin binding characteristics. A combination of molecular dynamics and NMR spectroscopy was used to examine potential polymer systems, in particular with the functional monomers methacrylic acid and 2-acrylamidopyridine. The synthesis of copolymers of ethylene dimethacrylate and one or both of these functional monomers was performed. A combination of radioligand binding studies and surface area analyses demonstrated the presence of selectivity in polymers prepared using methacrylic acid as the functional monomer. This was predicted by the molecular dynamics studies showing the power of this methodology as a prognostic tool for predicting the behavior of molecularly imprinted polymers.

  • 49. Williams, S. Kim R.
    et al.
    Caldwell, Karin D.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Physical Organic Chemistry.
    Field-flow fractionation2014In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 406, no 6, p. 1577-1578Article in journal (Other academic)
1 - 49 of 49
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