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Microchip electrophoresis utilizing an in situ photopolymerized Phos-tag binding polyacrylamide gel for specific entrapment and analysis of phosphorylated compounds
Analyst ( IF 3.6 ) Pub Date : 2017-08-15 00:00:00 , DOI: 10.1039/c7an00836h Sachio Yamamoto 1, 2, 3, 4 , Miyuki Himeno 1, 2, 3, 4 , Masaya Kobayashi 1, 2, 3, 4 , Miki Akamatsu 1, 2, 3, 4 , Ryosuke Satoh 1, 2, 3, 4 , Mitsuhiro Kinoshita 1, 2, 3, 4, 5 , Reiko Sugiura 1, 2, 3, 4, 5 , Shigeo Suzuki 1, 2, 3, 4, 5
Analyst ( IF 3.6 ) Pub Date : 2017-08-15 00:00:00 , DOI: 10.1039/c7an00836h Sachio Yamamoto 1, 2, 3, 4 , Miyuki Himeno 1, 2, 3, 4 , Masaya Kobayashi 1, 2, 3, 4 , Miki Akamatsu 1, 2, 3, 4 , Ryosuke Satoh 1, 2, 3, 4 , Mitsuhiro Kinoshita 1, 2, 3, 4, 5 , Reiko Sugiura 1, 2, 3, 4, 5 , Shigeo Suzuki 1, 2, 3, 4, 5
Affiliation
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A method was developed for the specific entrapment and separation of phosphorylated compounds using a Phos-tag polyacrylamide gel fabricated at the channel crossing point of a microfluidic electrophoresis chip. The channel intersection of the poly(methyl methacrylate)-made microchip was filled with a solution comprising acrylamide, N,N-methylene-bis-acrylamide, Phos-tag acrylamide, and 2,2′-azobis[2-methyl-N-(2-hydroxyethyl)propionamide], which functioned as a photocatalytic initiator. In situ polymerization at the channel crossing point was performed by irradiation with a UV LED laser beam. The fabricated Phos-tag gel (100 × 100 × 30 μm) contains ca. 20 fmol of the Phos-tag group and therefore could entrap phosphorylated compounds at the femtomolar level. The electrophoretically trapped phosphorylated compounds were released from the gel by switching the voltage to deliver high concentrations of phosphate and EDTA in a background electrolyte. The broad sample band eluted from the gel was effectively reconcentrated at the boundary of a pH junction generated by sodium ions delivered from the outlet reservoir. The reconcentrated sample components were then separated and fluorometrically detected at the end of the separation channel. Under the optimized conditions, the phosphorylated compounds were concentrated by a factor of 100-fold, and the peak resolution was comparable to that obtained by pinched injection. This method was successfully utilized to preconcentrate and analyze phosphorylated peptides in a complex peptide mixture.
中文翻译：
微芯片电泳利用原位光聚合的Phos-tag结合聚丙烯酰胺凝胶进行特定的捕获和磷酸化化合物的分析
开发了一种方法，该方法使用在微流体电泳芯片的通道交叉点制备的Phos-tag聚丙烯酰胺凝胶对磷酸化的化合物进行特定的捕获和分离。聚（甲基丙烯酸甲酯）制的微芯片的通道交叉点填充有包含丙烯酰胺，N，N - N-亚甲基-双丙烯酰胺，Phos-tag丙烯酰胺和2,2'-偶氮双[2-甲基-N-的溶液。 （2-羟乙基）丙酰胺]，起光催化引发剂的作用。通过用UV LED激光束照射在通道交叉点进行原位聚合。制成的Phos-tag凝胶（100×100×30μm）约含20 fmol的Phos-tag组，因此可以在飞摩尔水平截留磷酸化的化合物。通过切换电压以在背景电解质中输送高浓度的磷酸盐和EDTA，从凝胶中释放出电泳捕获的磷酸化化合物。从凝胶洗脱的宽样品带有效地重新浓缩在由出口储液器输送的钠离子产生的pH结的边界处。然后分离再浓缩的样品成分，并在分离通道的末端进行荧光检测。在优化的条件下，磷酸化的化合物浓缩了100倍，其峰分离度可与pin缩进样获得的峰分离度相媲美。
更新日期：2017-09-08
中文翻译：
微芯片电泳利用原位光聚合的Phos-tag结合聚丙烯酰胺凝胶进行特定的捕获和磷酸化化合物的分析
开发了一种方法，该方法使用在微流体电泳芯片的通道交叉点制备的Phos-tag聚丙烯酰胺凝胶对磷酸化的化合物进行特定的捕获和分离。聚（甲基丙烯酸甲酯）制的微芯片的通道交叉点填充有包含丙烯酰胺，N，N - N-亚甲基-双丙烯酰胺，Phos-tag丙烯酰胺和2,2'-偶氮双[2-甲基-N-的溶液。 （2-羟乙基）丙酰胺]，起光催化引发剂的作用。通过用UV LED激光束照射在通道交叉点进行原位聚合。制成的Phos-tag凝胶（100×100×30μm）约含20 fmol的Phos-tag组，因此可以在飞摩尔水平截留磷酸化的化合物。通过切换电压以在背景电解质中输送高浓度的磷酸盐和EDTA，从凝胶中释放出电泳捕获的磷酸化化合物。从凝胶洗脱的宽样品带有效地重新浓缩在由出口储液器输送的钠离子产生的pH结的边界处。然后分离再浓缩的样品成分，并在分离通道的末端进行荧光检测。在优化的条件下，磷酸化的化合物浓缩了100倍，其峰分离度可与pin缩进样获得的峰分离度相媲美。
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