Department of Chemistry, Division of Applied Physical Chemistry-Analytical Chemistry, KTH Royal Institute of Technology , 10044 Stockholm, Sweden.
Clinical Pharmacology Laboratory, Institute for Infectious Diseases, University of Bern , 3008 Bern, Switzerland.
Anal Chem. 2016 Oct 18;88(20):10044-10051. doi: 10.1021/acs.analchem.6b02324. Epub 2016 Sep 27.
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.
一种新的方法，用于通过等电聚焦（IEF）在 75nL 微通道中浓缩和纯化阿尔茨海默病相关的淀粉样β（Aβ）肽，结合微柱-基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）进行分析。提出了一种基于半开放式芯片的装置，由覆盖有液体氟碳盖的开放式微通道组成。IEF 在四种小的、化学上定义明确的两性载体（谷氨酸、天冬酰基-组氨酸（Asp-His）、环丝氨酸（cSer）和精氨酸）的混合物中进行，这些载体提供了一个逐步的 pH 梯度，专门用于在 cSer 和 Asp-His 之间聚焦具有等电点为 5.3 的 C 末端 Aβ肽。通过计算机模拟和直接从开放式微通道进行 MALDI-MS 分析，获得了关于聚焦动力学和 Aβ肽和其他化合物焦点位置的信息。使用建立的配置，通过直接从微通道中采样包含聚焦 Aβ肽的纳升级体积进行检测，然后将该体积沉积在带有微柱 MALDI 靶的芯片上。除了纯化之外，IEF 预浓缩还提供了至少 10 倍的 MALDI-MS 信号增强。在微通道中进行免疫沉淀和洗脱液浓缩后，IEF-微柱-MALDI-MS 被证明是一种适合检测人脑脊液和血浆中 Aβ肽的平台。
