Singapore Institute of Manufacturing Technology, 71 Nanyang Drive, Singapore 638075, Singapore.
J Chromatogr A. 2013 Aug 23;1304:220-6. doi: 10.1016/j.chroma.2013.06.073. Epub 2013 Jul 2.
To improve resolution of important minor proteins and eliminate time-consuming precipitation of major protein with associated analyte co-precipitation risk, a multi-dimension strategy is adopted in the 2D microchip-CE device to isolate major proteins on-chip, enrich minor proteins in capillary before their separation in CE for UV quantitation. A standard fluorescent protein mixture containing FITC-BSA, myoglobin and cytochrome as specific pI markers has prepared to demonstrate capability of the device to fractionate minor proteins by IEF. The results using a standard protein mixture with profile resembling infant milk formula show a complete isolation of high abundance proteins by a 2-min 1D IEF run. The subsequent t-ITP/CZE run by on-chip high voltage switching delivers a high stacking ratio, realizing 60 folds enrichment of isolated protein fractions. All five important functional proteins (LF, IgG, α-LA, β-LgA and β-LgB) known to fortify infant milk formula are isolated and determined using two consecutive t-ITP-CZE runs within a 18-min total assay time, a significant saving compared to several hours conventional pretreatment. For a 100g infant milk formula sample, working ranges of 20-8000mg, repeatability 3.8-5.3% and detection limits 2.3-10mg have been achieved to meet government regulations. Method reliability is established by 100% recoveries and agreeable results within expected ranges and labeled values. The capability of the device for field operation, rapid assay with quick results, label-free universal detection, simple operation by aqueous dissolution before injection, and the demanding matching in 2D separation based on isolated fractions at specified pI ranges, closely matched migration time and baseline-resolved peak shape makes the device a general tool to detect unknown proteins and determine known minor proteins in protein-rich samples with interfering constituents.
为了提高重要的次要蛋白质的分辨率并消除与主要蛋白质相关的分析物共沉淀风险的耗时沉淀，在二维微芯片 CE 装置中采用多维策略在芯片上分离主要蛋白质，在 CE 分离之前在毛细管中浓缩次要蛋白质以进行 UV 定量。使用含有 FITC-BSA、肌红蛋白和细胞色素作为特定 pI 标志物的标准荧光蛋白质混合物来证明该装置通过 IEF 分离次要蛋白质的能力。使用类似于婴儿配方奶粉的轮廓的标准蛋白质混合物的结果表明，通过 2 分钟的 1DIEF 运行完全分离高丰度蛋白质。随后通过芯片上高压切换进行 t-ITP/CZE 运行，实现高堆积比，实现分离蛋白质馏分的 60 倍富集。所有五种已知强化婴儿配方奶粉的重要功能蛋白（LF、IgG、α-LA、β-LgA 和 β-LgB）都被分离并通过两次连续的 t-ITP-CZE 运行在 18 分钟的总分析时间内确定，与传统预处理相比，这是一个显著的节省。对于 100g 婴儿配方奶粉样品，工作范围为 20-8000mg，重复性为 3.8-5.3%，检测限为 2.3-10mg，满足政府法规要求。通过 100%回收率和在预期范围内和标记值内的可接受结果来建立方法可靠性。该设备的现场操作能力、快速分析、快速结果、在注射前用水溶解的简单操作以及基于指定 pI 范围内分离馏分的二维分离的苛刻匹配、迁移时间接近和基线分辨的峰形使该设备成为一种通用工具，用于检测富含蛋白质的样品中的未知蛋白质和确定已知的次要蛋白质，其中含有干扰成分。
