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Off-line integration of CE and MALDI-MS using a closed-open-closed microchannel system
KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.ORCID iD: 0000-0003-3548-217X
KTH, School of Electrical Engineering (EES), Microsystem Technology.ORCID iD: 0000-0002-3996-9279
KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
KTH, School of Electrical Engineering (EES), Microsystem Technology.
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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.

Place, publisher, year, edition, pages
2007. Vol. 28, no 14, 2458-2465 p.
Keyword [en]
CE; MALDI-MS; off-line interface; silicon microcanal
National Category
Engineering and Technology
Identifiers
URN: urn:nbn:se:kth:diva-7875DOI: 10.1002/elps.200600735ISI: 000248390900017Scopus ID: 2-s2.0-34547442877OAI: oai:DiVA.org:kth-7875DiVA: diva2:13031
Note
QC 20100723Available from: 2008-01-09 Created: 2008-01-09 Last updated: 2011-11-21Bibliographically 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. MEMS Interfaces for Bioanalysis Systems
Open this publication in new window or tab >>MEMS Interfaces for Bioanalysis Systems
2008 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

This thesis deals with various aspects of using open microfluidic interfaces. Three specific areas of application are studied.

The first is air-to-liquid interfacing in biosensors with possibilities for component inte­gration. A micromachined interface for airborne sample-to-liquid and droplet-to-liquid adsorption is discussed. It enables a robust sheet liquid flow serving as adsorption site. The inter­face properties are presented. Along with the interface, a novel method and system for rapid detection of dust and vapour-based narcotics and explosives traces is introduced. The QCM sensor detection principle with antibody immunoassay is described. Having shown the working principles of molecular adsorption to liquid surface and molecular detection with QCM technology, an integrated device is introduced. Diffusion as an effective transport mechanism in this microfluidic device is discussed. By holding the two components (inter­face and QCM) together with a double-sided adhesive, anisotropically vertically conductive tape, we achieve three functions, namely fixation, electrical connection and liquid sealing. Finally, enhanced electrostatic trapping of small particles to the liquid interface is demon­strated.

The second area concerns open microfluidics for the integration of capillary electropho­resis and mass spectroscopy. A technique for hyphenation between CE and MALDI-MS is presented. Two closed fused-silica capillaries were connected via a silicon chip comprising an open microcanal. The influence of the capillary-to-microcanal connection is discussed, as well as a simple technique to control evaporation from the open microcanal.

The third area concerns microfluidics enabling studies of single cells in asymmetric en­vironments. Using extracellular matrix or synthetic gel-embedding cells in an assay chamber, cells thrive and proliferate. This makes it possible to carry out medium to long term cultiva­tion of cells in a more physiological, controlled 3D environment than in traditional 2D cul­tures. The gels are discussed in terms of handling as well as their properties. A gel and micro­fluidic device for three dimensional cell culture with microgradient environments is pre­sented. Finally, a method for studying cilia-forming cells in asymmetric microfluidic environments is presented. Bending the primary cilium with a fluid flow will give rise to a response, but sensitivity to flow direction has only been sparsely studied. Design considerations are presented and discussed.

Abstract [sv]

Den här avhandlingen diskuterar olika aspekter av den öppna gränsytan hos styckevis öppna mikrofluidiksystem. Tre specifika användningsområden har studerats.

Det första är gränsytan mellan luft och vätska i en biosensor och de användningsområ­den som finns här. Ett mikrofabricerat interface för adsorption av luftburna substanser samt dropp-absorption diskuteras. Här används en rörlig vätskeyta som adsorbtionsyta och dess egenskaper presenteras. En ny metod för sprängämnes- och narkotikadetektering med interfa­cet introduceras. QCM-tekniken i kombination med antikroppskemi beskrivs. En integrerad lösning med dessa tekniker introduceras där diffusion utgör en effektiv transportmekanism. Med en dubbelsidig ledande tejp hålls komponenterna ihop, tätas och förses med ström. Slut­ligen presenteras elektrostatisk infångning av partiklar där den ena elektroden utgörs av väts­keytan.

Det andra området berör ett öppet mikrofluidiksystem för integrering av kapillärelek­trofores och masspektrometri. Teknik för att koppla ihop CE och MALDI-MS presente­ras. Två glaskapillärer har kopplats ihop med ett kiselchip med en öppen mikrokanal. Kopp­lingen mellan kapillären och chippet diskuteras liksom en enkel teknik för att kontrol­lera avdunstningen från den öppna mikrokanalen.

Det tredje området diskuterar hur mikrofluidik möjliggör studier av cellulära reaktioner i asymmetriska miljöer. Med inbäddning av celler i extracellulär matris eller syntetisk gel fås fysiologiskt relevant lokal miljö för celltillväxt och celldelning. Detta möjliggör studier av cellutveckling och cellreaktioner under lång tid i faktisk 3D-miljö till skillnad från den nuva­rande etablerade 2D-miljön. Gelerna diskuteras ur en hanteringssynpunkt liksom utifrån sina egenskaper. Ett system för cellodling i 3D med gradi­entmiljö presenteras och diskuteras. Slutligen presenteras ett system för studier av ciliefor­mande cellers respons där asymmetriska flöden ger upphov till böjning av cilier. Olika de­signaspekter diskuteras.

Place, publisher, year, edition, pages
Stockholm: KTH, 2008. xii, 56 p.
Series
Trita-EE, ISSN 1653-5146 ; 2008:002
Keyword
microfluidic interfacing, microfluidics, µTAS, sample transfer, biosensor, electronic nose, surface tension, quartz crystal microbalance, QCM, narcotics detection
National Category
Control Engineering
Identifiers
urn:nbn:se:kth:diva-4609 (URN)978-91-7178-846-7 (ISBN)
Public defence
2008-02-08, Sal M3, KTH, Brinellvägen 64, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20100927Available from: 2008-01-18 Created: 2008-01-18 Last updated: 2010-09-27Bibliographically 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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