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Capillary electrophoresis separation and matrix-assisted laser desorption/ionization mass spectrometry characterization of bovine serum albumin fluorescein isothiocyanate conjugates
KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.ORCID iD: 0000-0003-3548-217X
KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
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2010 (English)In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 878, no 15-16, 1125-1134 p.Article in journal (Refereed) Published
Abstract [en]

A protocol using enzymatic digestion, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and capillary electrophoresis with laser induced fluorescence detection (CE-LIF) for the investigation of the binding of the fluorescent contact allergen fluorescein isothiocyanate (FITC) to the 66 kDa large protein bovine serum albumin (BSA), as a model system for protein-hapten binding in the skin, is presented. Mass spectra of BSA-FITC digestions, using trypsin and chymotrypsin, respectively, provided sequence coverage of 97%. To investigate the number of FITC-bound peptides using CE-LIF separation, three different buffer salts at four different pH levels were evaluated. The use of 20 mM sodium citrate pH 6.5 as well as 20 mM sodium phosphate pH 6.5 or pH 7.5 as background electrolyte revealed high numbers of peptides with at least one bound FITC. The effect of the electrolyte counter ion on MALDI-MS was investigated and was found to have effect on the MALDI spectra signal-to-noise (S/N) at 50 mM but not at 10 m M. Of the 60 theoretical FITC-binding sites in BSA this MALDI-MS protocol presents 30 defined. 28 possible and 2 non-binding sites for FITC. (C) 2010 Elsevier B.V. All rights reserved.

Place, publisher, year, edition, pages
2010. Vol. 878, no 15-16, 1125-1134 p.
Keyword [en]
Capillary electrophoresis, MALDI-TOF-MS, Contact allergy, Peptide-fluorescein isothiocyanate adducts, Bovine serum albumin
National Category
Analytical Chemistry
Identifiers
URN: urn:nbn:se:kth:diva-27396DOI: 10.1016/j.jchromb.2010.03.024ISI: 000277672000014OAI: oai:DiVA.org:kth-27396DiVA: diva2:377116
Note
QC 20101213Available from: 2010-12-13 Created: 2010-12-13 Last updated: 2017-12-11Bibliographically approved
In thesis
1. Improved techniques for CE and MALDI-MS including microfluidic hyphenations foranalysis of biomolecules
Open this publication in new window or tab >>Improved techniques for CE and MALDI-MS including microfluidic hyphenations foranalysis of biomolecules
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In this thesis, improved techniques for biomolecule analysis using capillary electrophoresis (CE) and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and hyphenations between those have been presented.A pre-concentration method which is possible to apply in both techniques, has also been investigated.

In this work the off-line MS mode has been used either in the form of fractionation (Paper I) or by incorporating the MALDI target in the CE separation system (Paper II).In Paper I, a protocol for CE-MALDI analysis of cyanogen bromide digested bacteriorhodopsin (BR) peptides as model integral membrane protein peptides were established. Also, an improved protocol for partially automated manufacturing of a concentration MALDI-target plate is presented. The design of the targets was suitable for the fractions from the CE.

A novel technique for the integration of CE to MALDI-MS using a closed-open-closed system is presented in Paper II, where the open part is a micro canal functioning as a MALDI target window. A protein separation was obtained and detected with MALDI-MS analysis in the micro canal.

A method has been developed for detection of monosaccharides originating from hydrolysis of a single wood fiber performed in a micro channel, with an incorporated electromigration pre-concentration step preceding CE analysis in Paper III. The pre-concentration showed to be highly complex due to the fact that several parameters are included that affecting each other.

In Paper IV a protocol using enzymatic digestion, MALDI-TOF-MS and CE with laser induced fluorescence (LIF) detection for the investigation of the degree of substitution of fluorescein isothiocyanate (FITC) to bovine serum albumin (BSA), as a contact allergen model system for protein-hapten binding in the skin, is presented. The intention of a further CE-MALDI hyphenation has been considered during the work.

In Paper V 2,6-dihydroxyacetophenone (DHAP) was investigated, showing promising MALDI-MS matrix properties for hydrophobic proteins and peptides. 2,5-dihydroxybenzoic acid (DHB) was undoubtedly the better matrix for the hydrophilic proteins, but its performance for the larger and hydrophobic peptides was not optimal. Consequently, DHAP can be used as a compliment matrix for improved analysis of hydrophobic analytes.

Place, publisher, year, edition, pages
Stockholm: KTH, 2011. Xii, 85 p.
Series
Trita-CHE-Report, ISSN 1654-1081 ; 2011.1
Keyword
Capillary electrophoresis, Hydrophobic peptides/proteins, Matrix-Assisted Laser Desorption Ionization Mass Spectrometry, Prestructured target, Off-line interface, Silicon micro canal, Matrix, Peptide-fluorescein isothiocyanate adducts, Bovine serum albumin, DHAP, Hyphenations, Hydrophobicity, Single wood fiber analysis, Pre-concentration, Concentration platform
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-27342 (URN)978-91-7415-808-3 (ISBN)
Public defence
2011-01-21, F3, Lindstedtsvägen 26, Stockholm, 10:00 (Swedish)
Opponent
Supervisors
Note
QC 20101214Available from: 2010-12-14 Created: 2010-12-10 Last updated: 2011-02-02Bibliographically approved

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Jacksén, JohanEmmer, Åsa

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