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通过毛细管电泳同时分析五种淀粉样肽作为阿尔茨海默病的潜在生物标志物。

Simultaneous analysis by capillary electrophoresis of five amyloid peptides as potential biomarkers of Alzheimer's disease.

作者信息

Verpillot Romain, Otto Markus, Klafki Hans, Taverna Myriam

机构信息

Univ Paris-Sud, JE 2495, Protéines et Nanotechnologies en Sciences Séparatives, Faculté de Pharmacie, 92296 Châtenay-Malabry, France.

出版信息

J Chromatogr A. 2008 Dec 19;1214(1-2):157-64. doi: 10.1016/j.chroma.2008.10.051. Epub 2008 Oct 18.

Abstract

We report here a CE method for the separation and quantitation of five amyloid peptides (Abeta1-42, 1-40, 1-39, 1-38, and 1-37) considered as potential biomarkers of Alzheimer's disease. These amyloid peptides have very similar structures. Sample preparation and storage conditions are critical parameters to ensure their solubility and to avoid the aggregation process in particular for Abeta1-42. Their solubility was found fully dependent on the NH(4)OH concentration that was employed initially to dissolve the lyophilized amyloid peptides. Conditions to achieve a full separation of these peptides were found using a dynamic coating with 1,4-diaminobutane (DAB). The linear decrease of their electrophoretic mobility highlighted an ion-pairing phenomenon between the peptides and DAB. The optimal background electrolyte was a 40 mM borate buffer, pH 9 containing 3 mM of DAB. Under these conditions, resolutions ranged from 1.3 to 2.4 with theoretical plates reaching 300,000. Under the retained conditions, we showed that adsorption of peptides to silica was negligible (recovery over 94.5%) and depletion effect of the background electrolyte was overcome. The method was finally validated in terms of linearity and repeatability and the limits of detection for the five Abeta peptides were estimated. The inter-day repeatability of the migration times was very satisfactory with RSDs less than 1.55%. The RSDs of the peak areas were below 5%. With this CE-UV method, limits of detection of the peptides ranged from 300 to 500 nM. We finally demonstrated that this method can be applied to real biological samples such as CSF.

摘要

我们在此报告一种毛细管电泳(CE)方法,用于分离和定量五种被视为阿尔茨海默病潜在生物标志物的淀粉样肽(β淀粉样蛋白1-42、1-40、1-39、1-38和1-37)。这些淀粉样肽结构非常相似。样品制备和储存条件是确保其溶解性并避免聚集过程的关键参数,尤其是对于β淀粉样蛋白1-42。发现它们的溶解性完全取决于最初用于溶解冻干淀粉样肽的氢氧化铵(NH₄OH)浓度。使用1,4-二氨基丁烷(DAB)进行动态涂层,找到了实现这些肽完全分离的条件。它们电泳迁移率的线性下降突出了肽与DAB之间的离子配对现象。最佳背景电解质是pH 9的40 mM硼酸盐缓冲液,含有3 mM的DAB。在这些条件下,分离度范围为1.3至2.4,理论塔板数达到300,000。在保留的条件下,我们表明肽对硅胶的吸附可忽略不计(回收率超过94.5%),并且克服了背景电解质的耗尽效应。该方法最终在线性和重复性方面得到验证,并估计了五种β淀粉样肽的检测限。迁移时间的日间重复性非常令人满意,相对标准偏差(RSD)小于1.55%。峰面积的RSD低于5%。使用这种CE-UV方法,肽的检测限范围为300至500 nM。我们最终证明该方法可应用于实际生物样品,如脑脊液(CSF)。

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