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Capillary electrophoretic separation and fractionation of hydrophobic peptides onto a pre-structured matrix assisted laser desorption/ionization target for mass spectrometric analysis
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.
KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.ORCID iD: 0000-0002-3444-9987
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.

Place, publisher, year, edition, pages
2006. Vol. 29, no 2, 288-295 p.
Keyword [en]
capillary electrophoresis, hydrophobic peptides, matrix-assisted laser desorption, ionization mass spectrometry, prestructured target
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:kth:diva-7874DOI: 10.1002/jssc.200500338ISI: 000235622100013Scopus ID: 2-s2.0-33644507426OAI: oai:DiVA.org:kth-7874DiVA: diva2:13030
Note
QC 20100901Available from: 2008-01-09 Created: 2008-01-09 Last updated: 2010-12-14Bibliographically 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
Opponent
Supervisors
Note
QC 20101109Available from: 2008-01-09 Created: 2008-01-09 Last updated: 2010-11-09Bibliographically approved
2. Improved Techniques for Protein Analysis Focusing on Membrane Proteins and Hydrophobic Peptides
Open this publication in new window or tab >>Improved Techniques for Protein Analysis Focusing on Membrane Proteins and Hydrophobic Peptides
2006 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

In this thesis, the vital cell functions performed by integral membrane proteins (IMPs) are briefly discussed. Such proteins are under-represented in most protein studies due to the hydrophobic nature of IMPs, which seriously complicate their solubilization, sample handling, preparation, separation and analysis. Conventional analytical techniques include for example matrix-assisted laser desorption/ionization mass spectrometry (MALDIMS), capillary electrophoresis (CE) and reversed phase high-performance liquid chromatography (RP-HPLC). Presented here are methods and protocols, which have been developed especially for IMP and hydrophobic peptide analysis, using the abovementioned techniques.

The fluorinated organic solvent hexafluoroisopropanol (HFIP) has previously been shown beneficial as an additive for solubilization of hydrophobic analytes, which are poorly soluble in commonly used organic solvents or water. In Papers I-IV, HFIP is successfully exploited as solvent for the investigated IMPs and peptides.

The simple fabrication and the focusing effect of a new structured MALDI target plate are presented in Paper I. This target plate contains concentrating sample spots, specifically designed to provide increased sensitivity for hydrophobic protein and peptide MALDI-MS analysis. When replacing a regular steel target with this new structured MALDI plate, more than a five-fold increase in average sensitivity is achieved for HFIP solubilized hydrophobic peptides. The full-length IMP bacteriorhodopsin (BR) and a cyanogen bromide digest thereof are used as model samples for the development of sample handling procedures in Paper II, and the peptides were used for evaluation of the MALDI-target plate in Paper I. Furthermore, the CE separation of the peptides, fractionation onto the structured MALDI plate and following MS analysis is presented in Paper III. Nine of the ten theoretical BR peptides were detected using this method.

A protocol for the purification and analysis of chloroplast membrane proteins from the green macroalga Ulva lactuca has been described in Paper IV. The highest protein yield was achieved when proteins were extracted in HFIP, directly from the chloroplasts. The MALDI-MS analysis of samples with and without previous RP-HPLC fractionation revealed proteins with molecular weights ranging between 1 and 376 kDa.

In Paper V, a closed-open-closed CE system is presented, containing an open microchannel for off-line MALDI detection. The electroosmotic flow and band broadening of this system has been evaluated.

Place, publisher, year, edition, pages
Stockholm: KTH, 2006. 68 p.
Keyword
Integral membrane proteins, Hydrophobic peptides, Sample handling, MALDI-TOF-MS, Structured sample support, CE, fraction collection, microchannel
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-617 (URN)91-7178-246-X (ISBN)
Public defence
2006-02-24, Sal E1, Lindstedtsvägen 3, Stockholm, 09:30
Opponent
Supervisors
Note
QC 20100916Available from: 2006-02-10 Created: 2006-02-10 Last updated: 2010-09-16Bibliographically approved
3. 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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