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Membrane protein and peptide sample handling for MS analysis using a structured MALDI target
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
2005 (English)In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Analytical and bioanalyltical chemistry, Vol. 381, no 1, 225-232 p.Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
2005. Vol. 381, no 1, 225-232 p.
Keyword [en]
structured sample support, MALDI-MS, hydrophobic peptides, integral membrane proteins, sample handling
National Category
Analytical Chemistry
Identifiers
URN: urn:nbn:se:kth:diva-9070DOI: 10.1007/s00216-004-2854-0ISI: 000226888300044Scopus ID: 2-s2.0-13844272668OAI: oai:DiVA.org:kth-9070DiVA: diva2:14625
Note
QC 20100916Available 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. 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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