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ELECTRO-OSMOTIC FLOW THROUGH CLOSED-OPEN-CLOSED MICROCHANNELS: AN APPROACH TO HYPHENATION OF CAPILLARY ELECTROPHORESIS AND MALDI
KTH, School of Electrical Engineering (EES), Microsystem Technology (Changed name 20121201).
KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry (closed 20110630).
KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry (closed 20110630).ORCID iD: 0000-0002-3444-9987
KTH, School of Electrical Engineering (EES), Microsystem Technology (Changed name 20121201).ORCID iD: 0000-0001-9552-4234
Show others and affiliations
2006 (English)In: 19th IEEE International Conference on Micro Electro Mechanical Systems (IEEE MEMS 2006), IEEE conference proceedings, 2006, 406-409 p.Conference paper, Published 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.

Place, publisher, year, edition, pages
IEEE conference proceedings, 2006. 406-409 p.
Series
Proceedings: IEEE Micro Electro Mechanical Systems Workshop, ISSN 1084-6999
Keyword [en]
Assisted-laser-desorption/ionization, mass-spectrometry, ms
National Category
Analytical Chemistry
Identifiers
URN: urn:nbn:se:kth:diva-9073DOI: 10.1109/MEMSYS.2006.1627822ISI: 000236994500102Scopus ID: 2-s2.0-33750139065ISBN: 978-078039475-9 (print)OAI: oai:DiVA.org:kth-9073DiVA: diva2:14628
Conference
19th IEEE International Conference on Micro Electro Mechanical Systems (IEEE MEMS 2006), Istanbul, TURKEY, 22-26 Jan, 2006
Note

QC 20100916

Available from: 2006-02-10 Created: 2006-02-10 Last updated: 2015-06-03Bibliographically approved
In thesis
1. Improved Techniques for Protein Analysis Focusing on Membrane Proteins and Hydrophobic Peptides
Open this publication in new window or tab >>Improved Techniques for Protein Analysis Focusing on Membrane Proteins and Hydrophobic Peptides
2006 (English)Doctoral 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.

Place, publisher, year, edition, pages
Stockholm: KTH, 2006. 68 p.
Keyword
Integral membrane proteins, Hydrophobic peptides, Sample handling, MALDI-TOF-MS, Structured sample support, CE, fraction collection, microchannel
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-617 (URN)91-7178-246-X (ISBN)
Public defence
2006-02-24, Sal E1, Lindstedtsvägen 3, Stockholm, 09:30
Opponent
Supervisors
Note
QC 20100916Available from: 2006-02-10 Created: 2006-02-10 Last updated: 2010-09-16Bibliographically approved

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Emmer, ÅsaStemme, GöranVan Der Wijngaart, Wouter

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