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Microfluidic Isoelectric Focusing of Amyloid Beta Peptides Followed by Micropillar-Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry
KTH, School of Chemical Science and Engineering (CHE), Chemistry, Applied Physical Chemistry. (Analytical Chemistry)
KTH, School of Chemical Science and Engineering (CHE), Chemistry, Applied Physical Chemistry. (Analytical Chemistry)ORCID iD: 0000-0003-3548-217X
KTH, School of Chemical Science and Engineering (CHE), Chemistry, Applied Physical Chemistry. (Analytical Chemistry)
University of Bern, Clinical Pharmacology Laboratory, Institute for Infectious Diseases.
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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: urn:nbn:se:kth:diva-193148DOI: 10.1021/acs.analchem.6b02324Scopus ID: 2-s2.0-84991795230OAI: oai:DiVA.org:kth-193148DiVA: diva2:998743
Note

QC 20160930

Available from: 2016-09-30 Created: 2016-09-30 Last updated: 2017-11-21Bibliographically approved
In thesis
1. Electrophoretic focusing in microchannels combined with mass spectrometry: Applications on amyloid beta peptides
Open this publication in new window or tab >>Electrophoretic focusing in microchannels combined with mass spectrometry: Applications on amyloid beta peptides
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
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:nbn:se:kth:diva-193134 (URN)978-91-7729-142-8 (ISBN)
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

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