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Membrane protein an...
Membrane protein and peptide sample handling for MS analysis using a structured MALDI target
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- Redeby, Theres (author)
- KTH,Analytisk kemi
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- Emmer, Åsa (author)
- KTH,Analytisk kemi
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(creator_code:org_t)
- 2004-12-22
- 2005
- English.
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In: Analytical and Bioanalytical Chemistry. - : Springer Science and Business Media LLC. - 1618-2642 .- 1618-2650. ; 381:1, s. 225-232
- Related links:
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https://urn.kb.se/re...
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https://doi.org/10.1...
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Abstract
Subject headings
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- 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.
Subject headings
- NATURVETENSKAP -- Kemi -- Analytisk kemi (hsv//swe)
- NATURAL SCIENCES -- Chemical Sciences -- Analytical Chemistry (hsv//eng)
Keyword
- structured sample support
- MALDI-MS
- hydrophobic peptides
- integral membrane proteins
- sample handling
- Analytical chemistry
- Analytisk kemi
Publication and Content Type
- ref (subject category)
- art (subject category)
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