Yu Cheng-Ju, Tseng Wei-Lung
Department of Chemistry, National SunYat-sen University, Kaohsiung, Taiwan.
Electrophoresis. 2006 Sep;27(18):3569-77. doi: 10.1002/elps.200600121.
We report an online concentration and separation method for basic proteins using poly(diallyldimethylammonium chloride) (PDDA) solutions in the presence of reversed EOF. Using a capillary dynamically coated with 2% PDDA containing 0.1 M NaCl and filled with 1.2% PDDA under neutral conditions (10 mM phosphate, pH 7.0), we have demonstrated the separation of six basic proteins with peak efficiencies ranging from 175 000 to 616 000 plates/m and RSDs of migration time less than 0.4%. Additionally, high-speed separation of six basic proteins (<7 min) was achieved using a short capillary filled with 0.6% PDDA solutions. Under injection of the large-volume sample (210 nL), the LODs at S/N of 3 for basic proteins are down to nanomolar range. For example, the LOD for lysozyme is 1.2 nM, which is a 260-fold sensitivity enhancement compared with conventional injection method. The proposed method has been applied to the stacking of lysozyme in human saliva samples. Without any pretreatment, we also demonstrated the capability of this method to detect low amounts of peptide samples through the stacking of tryptic peptide of myoglobin. The experimental results indicate that our proposed method has great potential for use in clinical diagnosis and proteomics applications.
我们报道了一种在反向电渗流存在下，使用聚二烯丙基二甲基氯化铵（PDDA）溶液对碱性蛋白质进行在线浓缩和分离的方法。使用在中性条件（10 mM磷酸盐，pH 7.0）下动态涂覆有含0.1 M NaCl的2% PDDA并填充1.2% PDDA的毛细管，我们已经证明了六种碱性蛋白质的分离，其峰效率范围为175000至616000塔板/米，迁移时间的相对标准偏差小于0.4%。此外，使用填充0.6% PDDA溶液的短毛细管实现了六种碱性蛋白质的高速分离（<7分钟）。在进样大体积样品（210 nL）时，碱性蛋白质在信噪比为3时的检测限低至纳摩尔范围。例如，溶菌酶的检测限为1.2 nM，与传统进样方法相比，灵敏度提高了260倍。所提出的方法已应用于人体唾液样品中溶菌酶的堆积。无需任何预处理，我们还通过肌红蛋白胰蛋白酶肽的堆积证明了该方法检测少量肽样品的能力。实验结果表明，我们提出的方法在临床诊断和蛋白质组学应用中具有巨大的潜力。
