Microfluidic Isoelectric Focusing of Amyloid Beta Peptides Followed by Micropillar-Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry

Abstract

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.

Recent developments in capillary and microchip electroseparations of peptides (2015–mid 2017)

“…Microchip IEF in a mixture of four small and chemically well‐defined amphoteric carriers, glutamic acid, dipeptide Asp‐His, cycloserine, and arginine, providing a stepwise pH gradient, was used for preconcentration and microisolation of Alzheimer's disease‐related Aβ peptides . The nanoliter volumes containing the focused peptides were deposited onto a chip with micropillar MALDI targets and analyzed by MALDI‐MS.…”

Proton Dynamics in Protein Mass Spectrometry

“…To address this issue, we combine here a multi-step computational protocol already established for a variety of proteins in the gas phase [22][23] with a quantum mechanics/molecular mechanics (QM/MM) approach. Our multi-scale approach is applied to a peptide fragment containing the 16 N-terminal residues of the ~40 amino acid-long amyloid- peptide (A(1-16)), a promising therapeutic target to reduce cognitive deficits for Alzheimer's disease patients, 31 which is broadly studied by MS characterization in aspects of structure 32 as well as their aggregation behaviors. [33][34] The maximum charge state (highest charge) of A (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16) is here predicted to be 4+ by our established hybrid Monte Carlo (MC)/MD-based protocol [22][23] (see Figure S1 in Supporting Information for details).…”

Go with the Flow—Microfluidics Approaches for Amyloid Research

“…The second methodology utilized isoelectric focusing( IEF) for both purification and pre-concentration of Ab polypeptides from CSF and bloodp lasma samples (Figure4). [93] IEF was performed using four amphoteric carriers to provide as tepwise pH gradient suited forf ocusing on the C-terminus of Ab polypeptides and was conducted in an open channel for easy sampling excess. In addition, the IEF was coupled with micropillarmatrix-assisted laser desorption ionization-time-of-flight-massspectrometry (MALDI-TOF-MASS) for detection.…”