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Simple fabrication of a structured matrix-assisted laser desorption/ionization target coating for increased sensitivity in mass spectrometric analysis of membrane proteins
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).
KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry (closed 20110630).ORCID iD: 0000-0002-3444-9987
2004 (English)In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 18, no 10, p. 1161-1166Article in journal (Refereed) Published
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

Place, publisher, year, edition, pages
2004. Vol. 18, no 10, p. 1161-1166
Keyword [en]
maldi-ms, sample supports, bacteriorhodopsin, identification, time, chromatography, proteomics, plates
National Category
Analytical Chemistry
Identifiers
URN: urn:nbn:se:kth:diva-9069DOI: 10.1002/rcm.1466ISI: 000221654900022Scopus ID: 2-s2.0-2542437060OAI: oai:DiVA.org:kth-9069DiVA, id: diva2:14624
Note

QC 20100916

Available from: 2006-02-10 Created: 2006-02-10 Last updated: 2017-12-14Bibliographically 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. p. 68
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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Publisher's full textScopushttp://www3.interscience.wiley.com/cgi-bin/abstract/108560811/ABSTRACT

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Emmer, Åsa

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