| 1. |
- Jacksén, Johan, et al.
(författare)
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Capillary electrophoresis separation and matrix-assisted laser desorption/ionization mass spectrometry characterization of bovine serum albumin fluorescein isothiocyanate conjugates
- 2010
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Ingår i: Journal of chromatography. B. - : Elsevier BV. - 1570-0232 .- 1873-376X. ; 878:15-16, s. 1125-1134
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Tidskriftsartikel (refereegranskat)abstract
- A protocol using enzymatic digestion, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and capillary electrophoresis with laser induced fluorescence detection (CE-LIF) for the investigation of the binding of the fluorescent contact allergen fluorescein isothiocyanate (FITC) to the 66 kDa large protein bovine serum albumin (BSA), as a model system for protein-hapten binding in the skin, is presented. Mass spectra of BSA-FITC digestions, using trypsin and chymotrypsin, respectively, provided sequence coverage of 97%. To investigate the number of FITC-bound peptides using CE-LIF separation, three different buffer salts at four different pH levels were evaluated. The use of 20 mM sodium citrate pH 6.5 as well as 20 mM sodium phosphate pH 6.5 or pH 7.5 as background electrolyte revealed high numbers of peptides with at least one bound FITC. The effect of the electrolyte counter ion on MALDI-MS was investigated and was found to have effect on the MALDI spectra signal-to-noise (S/N) at 50 mM but not at 10 m M. Of the 60 theoretical FITC-binding sites in BSA this MALDI-MS protocol presents 30 defined. 28 possible and 2 non-binding sites for FITC. (C) 2010 Elsevier B.V. All rights reserved.
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| 2. |
- Jacksén, Johan, et al.
(författare)
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Capillary electrophoretic separation and fractionation of hydrophobic peptides onto a pre-structured matrix assisted laser desorption/ionization target for mass spectrometric analysis
- 2006
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Ingår i: Journal of Separation Science. - : Wiley. - 1615-9306 .- 1615-9314. ; 29:2, s. 288-295
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Tidskriftsartikel (refereegranskat)abstract
- A CE separation of hydrophobic peptides followed by fractionation onto a prestructured MALDI target and off-line MS analysis was performed. An improved and partially automated manufacturing procedure of the previously described MALDI target is presented. This target is structurally coated with silicone and especially developed for hydrophobic peptides and proteins. Here, the target plate was designed specifically for the CE fraction collection. Different solvents were evaluated to meet the requirements of peptide solubility and compatibility to both the CE and MALDI methods and to the fractionation procedure. CE-MALDI-MS analysis of nine highly hydrophobic peptides from cyanogen bromide-digested bacteriorhodopsin is demonstrated.
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| 3. |
- Jacksén, Johan, et al.
(författare)
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Off-line integration of CE and MALDI-MS using a closed-open-closed microchannel system
- 2007
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 28:14, s. 2458-2465
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Tidskriftsartikel (refereegranskat)abstract
- In this work, a new technique for off-line hyphenation between CE and MALDI-MS is presented. Two closed fused-silica capillaries were connected via a silicon chip comprising an open microcanal. The EOF in the system was evaluated using mesityloxide or leucine-enkephalin as a sample and with a running buffer that rendered the analyte neutrally charged. Comparison was made between the EOF in a closed system (first capillary solely included in the electrical circuit) and in a closed-open system (first capillary and microcanal included in the electrical circuit). It was concluded that the experimental values of the EOF agreed with the theory. The influence of the capillary outer diameter on the peak dispersion was investigated using a closed-open-closed system (first capillary, microcanal and second capillary included in the electrical circuit). It was clearly seen that a capillary with 375 mu m od induced considerably higher peak dispersion than a 150 mu m od capillary, due to a larger liquid dead volume in the connection between the first capillary outlet and the microcanal. Mass spectrometric analysis has also been performed following CE separation runs in a closed-open-closed system with cytochrome c and lysozyme as model proteins. It was demonstrated that a signal distribution profile of the separated analytes could be recorded over a 30 mm long microcanal.
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| 4. |
- Johansson, Staffan, 1976, et al.
(författare)
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Mechanistic Proposal for the Formation of Specific Immunogenic Complexes via a Radical Pathway: A Key Step in Allergic Contact Dermatitis to Olefinic Hydroperoxides
- 2009
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Ingår i: Chem. Res. Toxicol.. - : American Chemical Society (ACS). - 0893-228X .- 1520-5010. ; 22:11, s. 1774-1781
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Tidskriftsartikel (refereegranskat)abstract
- The widespread use of scented products causes an increase of allergic contact dermatitis to fragrance compounds in Western countries today. Many fragrance compounds are prone to autoxidation, forming hydroperoxides as their primary oxidation products. Hydroperoxides are known to be strong allergens and to form specific immunogenic complexes. However, the mechanisms for the formation of the immunogenic complexes are largely unknown. We have investigated this mechanism for (5R)-5-isopropenyl-2-methyl-2-cyclohexene-1-hydroperoxide (Lim-2-OOH) by studying the formation of adducts in the reaction between this hydroperoxide and 5,10,15,20-tetraphenyl-21H,23H-porphine iron(III) chloride (Fe(III)TPPCl) in the presence of protected cysteine (NAc-Cys-OMe) or glutathione (GSH). Isolated adducts originate from the addition of the thiol group of NAc-Cys-OMe over the carbon−carbon double bonds of carvone. Furthermore, adducts between NAc-Cys-OMe and carveol as well as between GSH and carvone have been identified. The formation of these adducts most likely proceeds via the radical thiol−ene mechanism. The addition of a terpene moiety to cysteine offers an explanation of the specificity of the immune response to structurally different hydroperoxides. These results also explain the lack of cross-reactivity between carvone and Lim-2-OOH. In conclusion, we propose that immunogenic complexes of olefinic hydroperoxides can be formed via the radical thiol−ene mechanism. These complexes will be specific for the individual olefinic hydroperoxides due to the inclusion of a terpene moiety derived from the hydroperoxide.
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| 5. |
- Nilsson, Ulrika Nilsson, et al.
(författare)
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SPE and HPLC/UV of resin acids in colophonium-containing products.
- 2008
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Ingår i: Journal of separation science. - Weinheim, Fed. Rep. of Germany : Wiley. - 1615-9314 .- 1615-9306. ; 31:15, s. 2784-90
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Tidskriftsartikel (refereegranskat)abstract
- A new method, involving SPE and HPLC/UV diode-array detection (DAD), was developed for the quantification of colophonium components in different consumer products, such as cosmetics. Colophonium is a common cause of contact dermatitis since its components can oxidize into allergens on exposure to air. Three different resin acids were used as markers for native and oxidized colophonium, abietic acid (AbA), dehydroabietic acid (DeA), and 7-oxodehydroabietic acid (7-O-DeA). The SPE method, utilizing a mixed-mode hydrophobic and anion exchange retention mechanism, was shown to yield very clean extracts. The use of a urea-embedded C(12) HPLC stationary phase improved the separation of the resin acids compared to common C(18). Concentrations higher than 2 mg/g of both AbA and DeA were detected in wax strips. In this product also 7-O-DeA, a marker for oxidized colophonium, was detected at a level of 28 microg/g. The LODs were in the range of 7-19 microg/g and the LOQs 22-56 microg/g. The method is simple to use and can be applied on many types of technical products, not only cosmetics. For the first time, a method for technical products was developed, which separates AbA from pimaric acid.
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| 6. |
- Parmar, Varun, et al.
(författare)
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ELECTRO-OSMOTIC FLOW THROUGH CLOSED-OPEN-CLOSED MICROCHANNELS : AN APPROACH TO HYPHENATION OF CAPILLARY ELECTROPHORESIS AND MALDI
- 2006
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Ingår i: 19th IEEE International Conference on Micro Electro Mechanical Systems (IEEE MEMS 2006). - : IEEE conference proceedings. - 9780780394759 ; , s. 406-409
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Konferensbidrag (refereegranskat)abstract
- We suggest electro-osmotic driven flow (EOF) through closed-open-closed microchannels as a novel approach for spatial sample separation using capillary electrophoresis (CE) prior to matrix assisted laser desorption/ionization mass spectroscopy (MALDI-MS). For this purpose we built a system consisting of the series Coupling of a closed fused silica capillary for separation, a microfabricated open microcanal for future MS detection and a second closed fused silica capillary for downstream liquid collection. This work verifies the EOF transport of a peptide sample in such a system with low dispersion.
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| 7. |
- Redeby, Theres, et al.
(författare)
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A screening procedure for the solubilization of chloroplast membrane proteins from the marine green macroalga Ulva lactuca using RP-HPLC-MALDI-MS
- 2006
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Ingår i: International Journal of Biological Macromolecules. - : Elsevier BV. - 0141-8130 .- 1879-0003. ; 39:1-3, s. 29-36
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Tidskriftsartikel (refereegranskat)abstract
- A protocol for purification and analysis of chloroplast membrane proteins in the green macroalga Ulva lactuca has been developed, including reversed phase high performance liquid chromatography (RP-HPLC) and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Five different solvents were evaluated for extraction of membrane proteins by three methods. The highest protein yield was achieved when proteins were extracted directly from the chloroplasts using the solvent hexafluoroisopropanol. A range of proteins of increasing hydrophobicity was separated by HPLC. Analysis of both HPLC fractions and non-separated samples by MALDI-TOF-MS revealed proteins with molecular weights spanning between 1 and 376 kDa.
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| 8. |
- Redeby, Theres, 1973-
(författare)
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Improved Techniques for Protein Analysis Focusing on Membrane Proteins and Hydrophobic Peptides
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In this thesis, the vital cell functions performed by integral membrane proteins (IMPs) are briefly discussed. Such proteins are under-represented in most protein studies due to the hydrophobic nature of IMPs, which seriously complicate their solubilization, sample handling, preparation, separation and analysis. Conventional analytical techniques include for example matrix-assisted laser desorption/ionization mass spectrometry (MALDIMS), capillary electrophoresis (CE) and reversed phase high-performance liquid chromatography (RP-HPLC). Presented here are methods and protocols, which have been developed especially for IMP and hydrophobic peptide analysis, using the abovementioned techniques. The fluorinated organic solvent hexafluoroisopropanol (HFIP) has previously been shown beneficial as an additive for solubilization of hydrophobic analytes, which are poorly soluble in commonly used organic solvents or water. In Papers I-IV, HFIP is successfully exploited as solvent for the investigated IMPs and peptides. The simple fabrication and the focusing effect of a new structured MALDI target plate are presented in Paper I. This target plate contains concentrating sample spots, specifically designed to provide increased sensitivity for hydrophobic protein and peptide MALDI-MS analysis. When replacing a regular steel target with this new structured MALDI plate, more than a five-fold increase in average sensitivity is achieved for HFIP solubilized hydrophobic peptides. The full-length IMP bacteriorhodopsin (BR) and a cyanogen bromide digest thereof are used as model samples for the development of sample handling procedures in Paper II, and the peptides were used for evaluation of the MALDI-target plate in Paper I. Furthermore, the CE separation of the peptides, fractionation onto the structured MALDI plate and following MS analysis is presented in Paper III. Nine of the ten theoretical BR peptides were detected using this method. A protocol for the purification and analysis of chloroplast membrane proteins from the green macroalga Ulva lactuca has been described in Paper IV. The highest protein yield was achieved when proteins were extracted in HFIP, directly from the chloroplasts. The MALDI-MS analysis of samples with and without previous RP-HPLC fractionation revealed proteins with molecular weights ranging between 1 and 376 kDa. In Paper V, a closed-open-closed CE system is presented, containing an open microchannel for off-line MALDI detection. The electroosmotic flow and band broadening of this system has been evaluated.
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| 9. |
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| 10. |
- Redeby, Theres, et al.
(författare)
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Membrane protein and peptide sample handling for MS analysis using a structured MALDI target
- 2005
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Ingår i: Analytical and Bioanalytical Chemistry. - : Springer Science and Business Media LLC. - 1618-2642 .- 1618-2650. ; 381:1, s. 225-232
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Tidskriftsartikel (refereegranskat)abstract
- Different sample handling methods for hydrophobic proteins and peptides were evaluated in association with the utilization of a structured matrix-assisted laser/desorption ionization (MALDI) target for increased sensitivity. The fluorinated organic solvent hexafluoroisopropanol (HFIP) was used for the solubilization of both the full-length protein bacteriorhodopsin (BR) and a cyanogen bromide digest thereof, and compared to the performance of the non-ionic detergents octyl-beta-D-glucopyranoside (OG), dodecyl-beta-D-maltoside (DM), and Triton X-100. A concentrating effect was seen when using the structured MALDI plate for BR dissolved in all the different detergents, of which OG generated the best-quality spectra for the full-length integral membrane protein as well as for the hydrophobic peptides. However, the uneven analyte distribution obtained with the detergent preparations required selective and thus time-consuming acquisition of spectra. When instead HFIP was used as sample solvent, a tenfold increase in sensitivity was achieved for full-length BR. Addition of acids to the HFIP-solubilized sample, or to the MALDI matrix solution, improved the signals for a few of the peptides, while degrading the spectra of others. Consequently, the addition of acid could be used as a complementary sample preparation method for hydrophobic peptides. On-target washing to remove contaminants (e.g., salt) was performed, and a recrystallization protocol for signal improvement specifically suited for hydrophobic peptides is described. Results from digestion and solubilization in different micro centrifuge tubes were examined to determine the influence of different materials on the possible sample loss due to wall adhesion. Studies of sample solution storage times suggest immediate analysis after solubilization to obtain best results.
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