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Improved techniques for CE and MALDI-MS including microfluidic hyphenations foranalysis of biomolecules
KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.ORCID iD: 0000-0003-3548-217X
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 [en]
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: urn:nbn:se:kth:diva-27342ISBN: 978-91-7415-808-3 (print)OAI: oai:DiVA.org:kth-27342DiVA: diva2:376345
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
List of papers
1. Capillary electrophoretic separation and fractionation of hydrophobic peptides onto a pre-structured matrix assisted laser desorption/ionization target for mass spectrometric analysis
Open this publication in new window or tab >>Capillary electrophoretic separation and fractionation of hydrophobic peptides onto a pre-structured matrix assisted laser desorption/ionization target for mass spectrometric analysis
2006 (English)In: Journal of Separation Science, ISSN 1615-9306, E-ISSN 1615-9314, Vol. 29, no 2, 288-295 p.Article in journal (Refereed) Published
Abstract [en]

 A CE separation of hydrophobic peptides followed by fractionation onto a prestructured MALDI target and off-line MS analysis was performed. An improved and partially automated manufacturing procedure of the previously described MALDI target is presented. This target is structurally coated with silicone and especially developed for hydrophobic peptides and proteins. Here, the target plate was designed specifically for the CE fraction collection. Different solvents were evaluated to meet the requirements of peptide solubility and compatibility to both the CE and MALDI methods and to the fractionation procedure. CE-MALDI-MS analysis of nine highly hydrophobic peptides from cyanogen bromide-digested bacteriorhodopsin is demonstrated.

Keyword
capillary electrophoresis, hydrophobic peptides, matrix-assisted laser desorption, ionization mass spectrometry, prestructured target
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-7874 (URN)10.1002/jssc.200500338 (DOI)000235622100013 ()2-s2.0-33644507426 (Scopus ID)
Note
QC 20100901Available from: 2008-01-09 Created: 2008-01-09 Last updated: 2017-12-14Bibliographically approved
2. Off-line integration of CE and MALDI-MS using a closed-open-closed microchannel system
Open this publication in new window or tab >>Off-line integration of CE and MALDI-MS using a closed-open-closed microchannel system
Show others...
2007 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 28, no 14, 2458-2465 p.Article in journal (Refereed) Published
Abstract [en]

In this work, a new technique for off-line hyphenation between CE and MALDI-MS is presented. Two closed fused-silica capillaries were connected via a silicon chip comprising an open microcanal. The EOF in the system was evaluated using mesityloxide or leucine-enkephalin as a sample and with a running buffer that rendered the analyte neutrally charged. Comparison was made between the EOF in a closed system (first capillary solely included in the electrical circuit) and in a closed-open system (first capillary and microcanal included in the electrical circuit). It was concluded that the experimental values of the EOF agreed with the theory. The influence of the capillary outer diameter on the peak dispersion was investigated using a closed-open-closed system (first capillary, microcanal and second capillary included in the electrical circuit). It was clearly seen that a capillary with 375 mu m od induced considerably higher peak dispersion than a 150 mu m od capillary, due to a larger liquid dead volume in the connection between the first capillary outlet and the microcanal. Mass spectrometric analysis has also been performed following CE separation runs in a closed-open-closed system with cytochrome c and lysozyme as model proteins. It was demonstrated that a signal distribution profile of the separated analytes could be recorded over a 30 mm long microcanal.

Keyword
CE; MALDI-MS; off-line interface; silicon microcanal
National Category
Engineering and Technology
Identifiers
urn:nbn:se:kth:diva-7875 (URN)10.1002/elps.200600735 (DOI)000248390900017 ()2-s2.0-34547442877 (Scopus ID)
Note
QC 20100723Available from: 2008-01-09 Created: 2008-01-09 Last updated: 2017-12-14Bibliographically approved
3. CE analysis of single wood cells performing hydrolysis and preconcentration inopen micro channels
Open this publication in new window or tab >>CE analysis of single wood cells performing hydrolysis and preconcentration inopen micro channels
(English)Manuscript (preprint) (Other academic)
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-27609 (URN)
Note
QC 20101214Available from: 2010-12-14 Created: 2010-12-14 Last updated: 2010-12-14Bibliographically approved
4. Capillary electrophoresis separation and matrix-assisted laser desorption/ionization mass spectrometry characterization of bovine serum albumin fluorescein isothiocyanate conjugates
Open this publication in new window or tab >>Capillary electrophoresis separation and matrix-assisted laser desorption/ionization mass spectrometry characterization of bovine serum albumin fluorescein isothiocyanate conjugates
Show others...
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.

Keyword
Capillary electrophoresis, MALDI-TOF-MS, Contact allergy, Peptide-fluorescein isothiocyanate adducts, Bovine serum albumin
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-27396 (URN)10.1016/j.jchromb.2010.03.024 (DOI)000277672000014 ()
Note
QC 20101213Available from: 2010-12-13 Created: 2010-12-13 Last updated: 2017-12-11Bibliographically approved
5. Evaluation of 2,6-dihydroxyacetophenone as MALDI matrix for analysis ofhydrophobic proteins and peptides
Open this publication in new window or tab >>Evaluation of 2,6-dihydroxyacetophenone as MALDI matrix for analysis ofhydrophobic proteins and peptides
(English)Manuscript (preprint) (Other academic)
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-27610 (URN)
Note
QC 20101214Available from: 2010-12-14 Created: 2010-12-14 Last updated: 2010-12-14Bibliographically approved

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