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  • 1. Hellborg, Karin
    et al.
    Jacksén, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry.
    Skedung, Lisa
    KTH, School of Chemical Science and Engineering (CHE), Chemistry.
    Redeby, Therese
    KTH, School of Chemical Science and Engineering (CHE), Chemistry.
    Emmer, Åsa
    KTH, School of Chemical Science and Engineering (CHE), Chemistry.
    Evaluation of MALDI matrices and digestion methods aiming at MS analysis of hydrophobic proteins and peptidesManuscript (Other academic)
  • 2.
    Jacksén, Johan
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Frisk, Thomas
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Redeby, Theres
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Parmar, Varun
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    van der Wijngaart, Wouter
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Stemme, Göran
    KTH, School of Electrical Engineering (EES), Microsystem Technology.
    Emmer, Åsa
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Off-line integration of CE and MALDI-MS using a closed-open-closed microchannel system2007In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 28, no 14, p. 2458-2465Article in journal (Refereed)
    Abstract [en]

    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.

  • 3.
    Jacksén, Johan
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Redeby, Theres
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Emmer, Åsa
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Capillary electrophoretic separation and fractionation of hydrophobic peptides onto a pre-structured matrix assisted laser desorption/ionization target for mass spectrometric analysis2006In: Journal of Separation Science, ISSN 1615-9306, E-ISSN 1615-9314, Vol. 29, no 2, p. 288-295Article in journal (Refereed)
    Abstract [en]

     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.

  • 4.
    Parmar, Varun
    et al.
    KTH, School of Electrical Engineering (EES), Microsystem Technology (Changed name 20121201).
    Redeby, Theres
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry (closed 20110630).
    Emmer, Åsa
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry (closed 20110630).
    Stemme, Göran
    KTH, School of Electrical Engineering (EES), Microsystem Technology (Changed name 20121201).
    Van Der Wijngaart, Wouter
    KTH, School of Electrical Engineering (EES), Microsystem Technology (Changed name 20121201).
    ELECTRO-OSMOTIC FLOW THROUGH CLOSED-OPEN-CLOSED MICROCHANNELS: AN APPROACH TO HYPHENATION OF CAPILLARY ELECTROPHORESIS AND MALDI2006In: 19th IEEE International Conference on Micro Electro Mechanical Systems (IEEE MEMS 2006), IEEE conference proceedings, 2006, p. 406-409Conference paper (Refereed)
    Abstract [en]

    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.

  • 5.
    Redeby, Theres
    KTH, School of Chemical Science and Engineering (CHE), Chemistry.
    Improved Techniques for Protein Analysis Focusing on Membrane Proteins and Hydrophobic Peptides2006Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    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.

  • 6.
    Redeby, Theres
    KTH, Superseded Departments, Chemistry.
    Integral membrane protein MALDI-MS analysis - Sample handling and fabrication of a structured target2004Licentiate thesis, comprehensive summary (Other scientific)
  • 7.
    Redeby, Theres
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Carr, H.
    Björk, M.
    Emmer, Åsa
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    A screening procedure for the solubilization of chloroplast membrane proteins from the marine green macroalga Ulva lactuca using RP-HPLC-MALDI-MS2006In: International Journal of Biological Macromolecules, ISSN 0141-8130, E-ISSN 1879-0003, Vol. 39, no 1-3, p. 29-36Article in journal (Refereed)
    Abstract [en]

    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.

  • 8.
    Redeby, Theres
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Emmer, Åsa
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
    Membrane protein and peptide sample handling for MS analysis using a structured MALDI target2005In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Analytical and bioanalyltical chemistry, Vol. 381, no 1, p. 225-232Article in journal (Refereed)
    Abstract [en]

    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.

  • 9.
    Redeby, Theres
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry (closed 20110630).
    Roeraade, Johan
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry (closed 20110630).
    Emmer, Åsa
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry (closed 20110630).
    Simple fabrication of a structured matrix-assisted laser desorption/ionization target coating for increased sensitivity in mass spectrometric analysis of membrane proteins2004In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 18, no 10, p. 1161-1166Article in journal (Refereed)
    Abstract [en]

    A new prestructured target plate for matrix-assisted laser desorption/ionization (MALDI) was developed specifically for hydrophobic integral membrane proteins. This sample support contains predefined concentrating sample spots with a focusing effect on droplets with a high content of hexafluoroisopropanol (HFIP). This fluorinated organic solvent is advantageous for solubilizing hydrophobic proteins that are not soluble in water or the organic solvents normally used in sample preparation protocols for MALDI-MS. The prestructured plate was constructed by coating a regular steel plate with a thin layer of a silicone polymer, leaving sample spots of bare steel. Fabrication of the concentrating silicone structure was fast and very straightforward, without expensive or complicated equipment. Removing the layer, and thus regenerating the steel plate, was done by a simple washing procedure. The application and cleaning procedure are not constrained by a particular design of sample support or to any specific brand of mass spectrometer. When using the prestructured MALDI plate with HFIP as the sample solvent for 17 pmol of a cyanogen bromide digest of the highly hydrophobic membrane protein bacteriorhodopsin, an improved focusing effect and an increase of more than five-fold in average sensitivity were observed, compared with a regular steel target. Experimental results show a two-fold increase in average sensitivity when the new prestructured target plate was used, compared with a commercially available concentrating support

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