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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
KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.ORCID iD: 0000-0002-3444-9987
2006 (English)In: International Journal of Biological Macromolecules, ISSN 0141-8130, E-ISSN 1879-0003, Vol. 39, no 1-3, 29-36 p.Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
2006. Vol. 39, no 1-3, 29-36 p.
Keyword [en]
Ulva lactuca; HPLC; MALDI-TOF-MS
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:kth:diva-9072DOI: 10.1016/j.ijbiomac.2005.12.021ISI: 000239747300006Scopus ID: 2-s2.0-33746023964OAI: oai:DiVA.org:kth-9072DiVA: diva2:14627
Note
QC 20100825 Uppdaterad från accepted till published (20100825). Conference: European Workshop on Challenging Proteins. Paris, FRANCE. OCT 17-18, 2005Available from: 2006-02-10 Created: 2006-02-10 Last updated: 2011-09-27Bibliographically 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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