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Improved techniques for CE-MALDI-MS off-line coupling and MALDI-MS analysis of primarily hydrophobic proteins and peptides
KTH, School of Chemical Science and Engineering (CHE), Chemistry.ORCID iD: 0000-0003-3548-217X
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 [en]
Capillary electrophoresis, Hydrophobic peptides, Matrix-Assisted Laser Desorption Ionization Mass Spectrometry, Prestructured target, Off-line interface, Silicon microcanal, Matrix, Protein cleavage/digestion.
Keyword [sv]
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: urn:nbn:se:kth:diva-4599ISBN: 978-91-7178-836-8 (print)OAI: oai:DiVA.org:kth-4599DiVA: diva2:13033
Presentation
2008-01-24, E3, Huvudbyggnaden E, Osquarsbacke 14, KTH-Campus, 13:00
Opponent
Supervisors
Note
QC 20101109Available from: 2008-01-09 Created: 2008-01-09 Last updated: 2010-11-09Bibliographically 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: 2010-12-14Bibliographically 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: 2011-11-21Bibliographically approved
3. Evaluation of MALDI matrices and digestion methods aiming at MS analysis of hydrophobic proteins and peptides
Open this publication in new window or tab >>Evaluation of MALDI matrices and digestion methods aiming at MS analysis of hydrophobic proteins and peptides
Show others...
(English)Manuscript (Other academic)
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-7876 (URN)
Note
QC 20101109Available from: 2008-01-09 Created: 2008-01-09 Last updated: 2010-11-09Bibliographically approved

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