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Evaluation of MALDI matrices and digestion methods aiming at MS analysis of hydrophobic proteins and peptides
KTH, School of Chemical Science and Engineering (CHE), Chemistry.ORCID iD: 0000-0003-3548-217X
KTH, School of Chemical Science and Engineering (CHE), Chemistry.
KTH, School of Chemical Science and Engineering (CHE), Chemistry.
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(English)Manuscript (Other academic)
National Category
Analytical Chemistry
Identifiers
URN: urn:nbn:se:kth:diva-7876OAI: oai:DiVA.org:kth-7876DiVA: diva2:13032
Note
QC 20101109Available from: 2008-01-09 Created: 2008-01-09 Last updated: 2010-11-09Bibliographically approved
In thesis
1. Improved techniques for CE-MALDI-MS off-line coupling and MALDI-MS analysis of primarily hydrophobic proteins and peptides
Open this publication in new window or tab >>Improved techniques for CE-MALDI-MS off-line coupling and MALDI-MS analysis of primarily hydrophobic proteins and peptides
2007 (English)Licentiate thesis, comprehensive summary (Other scientific)
Abstract [en]

Due to the hydrophobic nature of integral membrane proteins (IMP) they give rise to several difficulties concerning handling and analysis, which is not the case for the most water soluble proteins. New analysis methods are needed, where the insolubility problems of the hydrophobic proteins due to aggregation and adhesion are tackled. Those problems also affect digestion performance and equipment compatibility for the analysis.

Protocols for analysis and separation specified for IMP are presented in Paper I and III.

The instrumentation used in this work was capillary electrophoresis (CE) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Both instruments are suitable for peptide/proteins analysis.

In Paper I, protocols for a CE separation of bacteriorhodopsin (BR) peptides as model IMP peptides are established. Also, a partially automated manufacturing procedure of a concentration MALDI-target is presented, suitable for fractions from CE. The MS analysis detected 9 out of 10 cyanogen bromide (CNBr) digested BR peptides. A novel technique for the off-line integration of CE to MALDI-MS using a closed-open-closed system is presented in Paper II, where the open part is a microcanal functioning as a MALDI target window. Investigation of the microcanal electro-osmotic flow (EOF) properties and band broadening characteristics was performed. A protein separation was obtained and detected with MALDI-MS analysis in the microcanal. Different protein digestion methods were evaluated using BR in Paper III through MALDI-MS. Several digestion methods as well as MS media were investigated alongside different MALDI matrices. For example, matrices as the hydrophobic 2,6-dihydroxyacetophenone (DHAP) and 2-Hydroxy-3-methoxybenzoic acid (2H3MBA) or 2-Hydroxy-5-methoxybenzoic acid (2H5MBA) mixed with DHB, appeared to be promising matrices for analysis of BR.

Abstract [sv]

Med anledning av integrala membranproteiners (IMP) hydrofoba egenskaper uppstår flera svårigheter vid hantering och analys av IMP, vilket inte är fallet för vattenlösliga proteiner. Nya analysmetoder krävs, som löser löslighetsproblemen för de hydrofoba proteinerna som tex flockning och adsorbtion. Dessa problem påverkar även klyvningsgrad och kompatibilitet med analysutrustningen.

I Artikel I och Artikel III presenteras protokoll för analys och separation specifikt för IMP. Instrumenteringen som har använts i detta arbete är kapillärelektrofores (CE) och matris-assisterad laserdesorptions-joniserings-masspektrometri (MALDI-MS). Båda instrumenten är lämpade för peptid/protein analyser.

I Artikel I, presenteras protokoll för en CE separation av peptider från bacteriorhodopsin (BR), som användes som modellpeptider för IMP. En delvis automatiserat tillverkningsprocedur för en koncentrerande MALDI-platta, som är anpassad för CE fraktionerna beskrivs också. MS-analysen detekterade 9 av 10 BR-peptider från cyanobromid-klyvning (CNBr). En ny teknik för off line-integrering av CE till MALDI-MS genom ett slutet-öppet-slutet system presenteras i Artikel II, där den öppna delen är en mikrokanal som fungerar som detektionsfönster i MALDI. Undersökning av mikrokanalens egenskaper som tex det elektroosmotiska flödet (EOF) och bandbreddningen utvärderades. En proteinseparation genomfördes och detekterades med MALDI–MS i mikrokanalen. Olika proteinklyvningsmetoder för BR undersöktes i Artikel III med MALDI-MS. Flera proteinklyvningsmetoder samt MS-medier utvärderades tillsammans med olika MALDI-matriser. Den hydrofoba matrisen 2,6-dihydroxyacetophenone (DHAP) och 2-Hydroxy-3-methoxybenzoic acid (2H3MBA) eller 2-Hydroxy-5-methoxybenzoic acid (2H5MBA) blandade med DHB, visade sig exempelvis vara lovande matriser för BR-analyser.

Place, publisher, year, edition, pages
Stockholm: KTH, 2007. viii, 42 p.
Series
Trita-CHE-Report, ISSN 1654-1081 ; 2007:85
Keyword
Capillary electrophoresis, Hydrophobic peptides, Matrix-Assisted Laser Desorption Ionization Mass Spectrometry, Prestructured target, Off-line interface, Silicon microcanal, Matrix, Protein cleavage/digestion., Kapillärelektrofores, Hydrofoba peptider, Matris-assisterad laserdesorptions-joniserings-masspektrometri, Strukturbelagd provplatta, Off-line interface, Mikrokanal i kisel, Matris, Proteinklyvning/-spjälkning.
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-4599 (URN)978-91-7178-836-8 (ISBN)
Presentation
2008-01-24, E3, Huvudbyggnaden E, Osquarsbacke 14, KTH-Campus, 13:00
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Note
QC 20101109Available from: 2008-01-09 Created: 2008-01-09 Last updated: 2010-11-09Bibliographically approved

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