Microfluidic Isoelectric Focusing of Amyloid Beta Peptides Followed by Micropillar-Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry.
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.
Department of Chemistry, Division of Applied Physical Chemistry-Analytical Chemistry, KTH Royal Institute of Technology , 10044 Stockholm, Sweden.
Saara
S
Mikkonen
Department of Chemistry, Division of Applied Physical Chemistry-Analytical Chemistry, KTH Royal Institute of Technology , 10044 Stockholm, Sweden.
Johan
J
Jacksén
Department of Chemistry, Division of Applied Physical Chemistry-Analytical Chemistry, KTH Royal Institute of Technology , 10044 Stockholm, Sweden.
Johan
J
Roeraade
Clinical Pharmacology Laboratory, Institute for Infectious Diseases, University of Bern , 3008 Bern, Switzerland.
Wolfgang
W
Thormann
Department of Chemistry, Division of Applied Physical Chemistry-Analytical Chemistry, KTH Royal Institute of Technology , 10044 Stockholm, Sweden.
Åsa
Å
Emmer
D016229:Amyloid beta-Peptides / Q000097:blood* / Q000134:cerebrospinal fluid*; D003198:Computer Simulation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000379:methods*; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D016229:Amyloid beta-Peptides