Fluorescent derivatization method of proteins for characterization by capillary elect rophoresis-sodium dodecyl sulfate with laser-induced fluorescence detection
Fluorescent derivatization method of proteins for characterization by capillary elect rophoresis-sodium dodecyl sulfate with laser-induced fluorescence detection
复制标题
DOI：
10.1021/ac0705521
复制
发表时间：
2007-08-01
影响因子：
7.4
通讯作者：
Balland, Alain
中科院分区：
文献类型：
--
作者：
Michels, David A.;Brady, Lowell J.;Balland, Alain
建立了一种快速、高效的毛细管电泳-十二烷基硫酸钠（CE-SDS）激光诱导荧光检测蛋白质的方法。该CE-SDS法开发用于重组单克隆抗体（rMAb）的纯度测定。在本试验中，在存在亲核试剂（CN-）的情况下，用荧光试剂3-（2-呋喃甲酰基）-喹啉-2-甲醛（FQ）衍生rMAb，亲核试剂仅在与蛋白质共价结合时发出荧光。因此，标记后的纯化不需要去除未反应的试剂。将蛋白质在75 ° C下孵育5分钟以促进变性和标记。对于非还原制备，在pH 6.5下用50：1的染料与蛋白质（DIP）摩尔比标记rMAb，其形成具有6 × 4 FQ标记的缀合物。对于还原制备，在pH 9.3下以10：1的DIP摩尔比标记rMAb，其产生掺入有3 × 2 FQ标记物的轻链缀合物。使用烷基化试剂时，不存在标记伪影，如片段化或聚集。这种有效的标记方案产生的检测限低至10 ng/mL的荧光标记的rMAbs相比，其他标记策略，标记蛋白质与FQ具有速度，易用性和强大的定量的优势。
A fast and improved sample preparation scheme was developed for protein analysis using capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) with laser-induced fluorescence detection. This CE-SDS method was developed as a purity assay for recombinant monoclonal antibodies (rMAbs). In this assay, rMAbs are derivatized with the fluorogenic reagent 3-(2-furoyl)-quinoline-2-carboxaldehyde (FQ) in the presence of a nucleophile (CN-), which fluoresces only upon covalent binding to the protein. Purification after labeling is therefore not necessary to remove unreacted reagents. Proteins are incubated at 75 degrees C for 5 min to facilitate denaturation and labeling. For nonreduced preparation, rMAbs are labeled at pH 6.5 with a dye-to-protein (DIP) molar ratio of 50: 1, which forms conjugates having 6 4 FQ labels. For reduced preparation, rMAbs are labeled at pH 9.3 with a DIP molar ratio of 10: 1, which generates light chain conjugates incorporated with 3 2 FQ labels. Labeling artifacts such as fragmentation or aggregation are absent with use of alkylation reagents. This efficient labeling scheme generates detection limits for FQ-labeled rMAbs as low as 10 ng/mL In comparison to other labeling strategies, labeling proteins with FQ has the advantage of speed, ease of use, and robust quantification.