Yamamoto Sachio, Yano Shoko, Kinoshita Mitsuhiro, Suzuki Shigeo
Faculty of Pharmacy, Kindai University, 3-4-1 Kowakae, Higashiosaka 577-8502, Osaka, Japan.
Antiaging Center, Kindai University, 3-4-1 Kowakae, Higashiosaka 577-8502, Osaka, Japan.
Gels. 2021 Dec 16;7(4):268. doi: 10.3390/gels7040268.
An improved method for the online preconcentration, derivatization, and separation of phosphorylated compounds was developed based on the affinity of a Phos-tag acrylamide gel formed at the intersection of a polydimethylsiloxane/glass multichannel microfluidic chip toward these compounds. The acrylamide solution comprised Phos-tag acrylamide, acrylamide, and -methylene-bis-acrylamide, while 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide] was used as a photocatalytic initiator. The Phos-tag acrylamide gel was formed around the channel crossing point via irradiation with a 365 nm LED laser. The phosphorylated peptides were specifically concentrated in the Phos-tag acrylamide gel by applying a voltage across the gel plug. After entrapment of the phosphorylated compounds in the Phos-tag acrylamide gel, 5-(4,6-dichlorotriazinyl)aminofluorescein (DTAF) was introduced to the gel for online derivatization of the concentrated phosphorylated compounds. The online derivatized DTAF-labeled phosphorylated compounds were eluted by delivering a complex of phosphate ions and ethylenediamine tetraacetic acid as the separation buffer. This method enabled sensitive analysis of the phosphorylated peptides.
基于聚二甲基硅氧烷/玻璃多通道微流控芯片交叉点处形成的Phos-tag丙烯酰胺凝胶对磷酸化化合物的亲和力，开发了一种用于在线预浓缩、衍生化和分离磷酸化化合物的改进方法。丙烯酰胺溶液包含Phos-tag丙烯酰胺、丙烯酰胺和N,N'-亚甲基双丙烯酰胺，同时使用2,2'-偶氮二[2-甲基-N-(2-羟乙基)丙酰胺]作为光催化引发剂。通过用365 nm LED激光照射，在通道交叉点周围形成Phos-tag丙烯酰胺凝胶。通过在凝胶塞两端施加电压，将磷酸化肽特异性浓缩在Phos-tag丙烯酰胺凝胶中。将磷酸化化合物截留在Phos-tag丙烯酰胺凝胶中后，将5-(4,6-二氯三嗪基)氨基荧光素（DTAF）引入凝胶中，对浓缩的磷酸化化合物进行在线衍生化。通过输送磷酸根离子和乙二胺四乙酸的络合物作为分离缓冲液，洗脱在线衍生化的DTAF标记的磷酸化化合物。该方法能够对磷酸化肽进行灵敏分析。
