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Electrophoretic focusing in microchannels combined with mass spectrometry: Applications on amyloid beta peptides
KTH, School of Chemical Science and Engineering (CHE), Chemistry, Applied Physical Chemistry. (Analytical Chemistry)
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Analysis of low-abundance components in small samples remains a challenge within bioanalytical chemistry, and new techniques for sample pretreatments followed by sensitive and informative detection are required. In this thesis, procedures for preconcentration and separation of proteins and peptides in open microchannels fabricated on silicon microchips are presented. Analyte electromigration was induced by applying a voltage along the channel length, and detection was performed either by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) within the open channel, or by sampling a nL fraction containing the preconcentrated analytes from the channel for subsequent nano-electrospray ionization- (nESI-) or MALDI-MS. Utilizing solvent evaporation from the open system during sample supply, sample volumes exceeding the 25-75 nL channel volume could be analyzed. For preconcentration/separation of components in the discrete channel volume a lid of inert fluorocarbon liquid was used for evaporation control.

In Papers I and II, aqueous, carrier-free solutions of proteins and peptides were analyzed, and the method was successfully applied for fast and simple preconcentration of amyloid beta (Aβ) peptides, related to Alzheimer’s disease.

The impact of possible impurities in the analysis of carrier-free solutions was investigated in Paper III with the 1D simulation software GENTRANS, and a method for open-channel isoelectric focusing in a tailor-made pH gradient was developed. The latter approach was used in Paper IV for preconcentration and purification of Aβ peptides after immunoprecipitation from cerebrospinal fluid and blood plasma, followed by MALDI-MS from a micropillar chip.

Paper V includes simulations of an isotachophoretic strategy for selective enrichment of Aβ peptides. GENTRANS simulations were used to select the electrolyte composition, and 2D simulations in a geometry suitable for on-chip implementation were performed using COMSOL Multiphysics.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2016. , 55 p.
Series
TRITA-CHE-Report, ISSN 1654-1081 ; 2016:36
Keyword [en]
amyloid beta, computer simulation, electrophoresis, electrospray ionization, isoelectric focusing, isotachophoresis, MALDI, mass spectrometry, microchannel, microchip, nano-electrospray ionization, preconcentration, separation
National Category
Analytical Chemistry
Research subject
Chemistry
Identifiers
URN: urn:nbn:se:kth:diva-193134ISBN: 978-91-7729-142-8 (print)OAI: oai:DiVA.org:kth-193134DiVA: diva2:998789
Public defence
2016-11-04, F3, Lindstedtsvägen 26, 10:00 (English)
Opponent
Supervisors
Note

QC 20160930

Available from: 2016-09-30 Created: 2016-09-29 Last updated: 2016-09-30Bibliographically approved
List of papers
1. Sample preconcentration in open microchannels combined with MALDI-MS
Open this publication in new window or tab >>Sample preconcentration in open microchannels combined with MALDI-MS
2012 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 33, no 22, 3343-3350 p.Article in journal (Refereed) Published
Abstract [en]

In this work, a method for preconcentrating samples in 1 cm long, 50-150 μm wide open microchannels is presented. Platinum electrodes were positioned at the channel ends, voltage was applied, and charged analyte was preconcentrated at the oppositely charged side during continuous supply of sample. The preconcentration was initially studied in a closed system, where an influence on the analyte position from a pH gradient, generated by water electrolysis, was observed. In the open channel, the analyte distribution after preconcentration was evaluated using MALDI-MS with the channel as MALDI target. MALDI matrix was applied with an airbrush or by electrospray matrix deposition and by using the latter technique higher degrees of crystallization in the channels were obtained. After preconcentrating a 1 nM cytochrome c solution for 5 min, corresponding to a supplied amount of 1.25 fmol, a signal on the cathodic channel end could be detected. When a solution of cytochrome c trypsin digest was supplied, the peptides were preconcentrated at different positions along the channel depending on their charge.

Keyword
Electrospray deposition, MALDI-MS, Preconcentration, Silicon microchip
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-108041 (URN)10.1002/elps.201200129 (DOI)000311303100013 ()2-s2.0-84869782576 (Scopus ID)
Funder
Swedish Research Council
Note

QC 20121220

Available from: 2012-12-20 Created: 2012-12-19 Last updated: 2017-12-06Bibliographically approved
2. Mass spectrometric analysis of nanoscale sample volumes extracted from open microchannels after sample preconcentration applied on amyloid beta peptides
Open this publication in new window or tab >>Mass spectrometric analysis of nanoscale sample volumes extracted from open microchannels after sample preconcentration applied on amyloid beta peptides
2014 (English)In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 406, no 14, 3521-3524 p.Article in journal (Refereed) Published
Abstract [en]

A new instrumental concept for extraction of nanovolumes from open microchannels (dimensions 150 mu m x 50 mu m, length 10 mm) manufactured on silicon microchips has been used in combination with a previously developed method for preconcentrating proteins and peptides in the open channels through electromigration. The extracted nanovolumes were further analyzed using nanoelectrospray ionization (nESI) or matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) directly or with subsequent enzymatic protein digestion in a nanodroplet prior to the MS analysis. Preconcentration of the samples resulted in a 15-fold sensitivity increase in nESI for a neurotensin solution, and using MALDI-MS, amyloid beta (A beta) peptides could be detected in concentrations down to 1 nM. The method was also successfully applied for detection of cell culture A beta.

Keyword
Mass spectrometry, Microchannel, Preconcentration, Amyloid beta, MALDI, Nano-ESI
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-147047 (URN)10.1007/s00216-014-7781-0 (DOI)000336261800030 ()2-s2.0-84901603229 (Scopus ID)
Funder
Swedish Research Council, 621-2009-4095
Note

QC 20140624

Available from: 2014-06-24 Created: 2014-06-23 Last updated: 2017-12-05Bibliographically approved
3. Computer simulations of sample preconcentration in carrier-free systems and isoelectric focusing in microchannels using simple ampholytes
Open this publication in new window or tab >>Computer simulations of sample preconcentration in carrier-free systems and isoelectric focusing in microchannels using simple ampholytes
2015 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 36, no 19, 2386-2395 p.Article in journal (Refereed) Published
Abstract [en]

In this work, electrophoretic preconcentration of protein and peptide samples in microchannels was studied theoretically using the 1D dynamic simulator GENTRANS, and experimentally combined with MS. In all configurations studied, the sample was uniformly distributed throughout the channel before power application, and driving electrodes were used as microchannel ends. In the first part, previously obtained experimental results from carrier-free systems are compared to simulation results, and the effects of atmospheric carbon dioxide and impurities in the sample solution are examined. Simulation provided insight into the dynamics of the transport of all components under the applied electric field and revealed the formation of a pure water zone in the channel center. In the second part, the use of an IEF procedure with simple well defined amphoteric carrier components, i.e. amino acids, for concentration and fractionation of peptides was investigated. By performing simulations a qualitative description of the analyte behavior in this system was obtained. Neurotensin and [Glu1]-Fibrinopeptide B were separated by IEF in microchannels featuring a liquid lid for simple sample handling and placement of the driving electrodes. Component distributions in the channel were detected using MALDI- and nano-ESI-MS and data were in agreement with those obtained by simulation. Dynamic simulations are demonstrated to represent an effective tool to investigate the electrophoretic behavior of all components in the microchannel.

Place, publisher, year, edition, pages
Wiley-VCH Verlagsgesellschaft, 2015
Keyword
Isoelectric focusing, Mass spectrometry, Microchip, Preconcentration, Simulation
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-175914 (URN)10.1002/elps.201500120 (DOI)000362187700003 ()26036978 (PubMedID)2-s2.0-84957847418 (Scopus ID)
Funder
Swedish Research Council
Note

QC 20151104

Available from: 2015-11-04 Created: 2015-10-26 Last updated: 2017-12-01Bibliographically approved
4. Microfluidic Isoelectric Focusing of Amyloid Beta Peptides Followed by Micropillar-Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry
Open this publication in new window or tab >>Microfluidic Isoelectric Focusing of Amyloid Beta Peptides Followed by Micropillar-Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry
Show others...
2016 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882Article in journal (Refereed) Epub ahead of print
Abstract [en]

A novel method for preconcentration and purification of the Alzheimer’s disease related amyloid beta (Aβ) peptides by isoelectric focusing (IEF) in 75 nL microchannels combined with their analysis by micropillar-matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) is presented. A semiopen chip-based setup, consisting of open microchannels covered by a lid of a liquid fluorocarbon, was used. IEF was performed in a mixture of four small and chemically well-defined amphoteric carriers, glutamic acid, aspartyl-histidine (Asp-His), cycloserine (cSer), and arginine, which provided a stepwise pH gradient tailored for focusing of the C-terminal Aβ peptides with a pI of 5.3 in the boundary between cSer and Asp-His. Information about the focusing dynamics and location of the foci of Aβ peptides and other compounds was obtained using computer simulation and by performing MALDI-MS analysis directly from the open microchannel. With the established configuration, detection was performed by direct sampling of a nanoliter volume containing the focused Aβ peptides from the microchannel, followed by deposition of this volume onto a chip with micropillar MALDI targets. In addition to purification, IEF preconcentration provides at least a 10-fold increase of the MALDI-MS-signal. After immunoprecipitation and concentration of the eluate in the microchannel, IEF-micropillar-MALDI-MS is demonstrated to be a suitable platform for detection of Aβ peptides in human cerebrospinal fluid as well as in blood plasma.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2016
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-193148 (URN)10.1021/acs.analchem.6b02324 (DOI)2-s2.0-84991795230 (Scopus ID)
Note

QC 20160930

Available from: 2016-09-30 Created: 2016-09-30 Last updated: 2017-11-21Bibliographically approved
5. Selective enrichment of amyloid beta peptides using isotachophoresis
Open this publication in new window or tab >>Selective enrichment of amyloid beta peptides using isotachophoresis
(English)Manuscript (preprint) (Other academic)
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-193149 (URN)
Note

QC 20160930

Available from: 2016-09-30 Created: 2016-09-30 Last updated: 2016-09-30Bibliographically approved

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