NiⅡ-NTA修饰的银纳米粒/多孔硅芯片在线分离组氨酸标记蛋白和MALDI-TOF质谱检测

摘要

An affinity chip was developed via self-assembly of 4-aminothiophenol onto silver nanoparticles (AgNPs)/porous silicon (PSi) chip and chemical conversion of amino groups to Ni Ⅱ-nitrilotriacetic acid (Ni Ⅱ-NTA) termini.The NiⅡ-NTA modified chip was applied to separate and preconcentrate histidine-tagged (his-tagged) fusion proteins,thioredoxin-urodilatin and the lysate of small ubiquitin-related modifier (SUMO)-hu-aprotinin,in a buffer solution containing high levels of salts and solubilizing agents.The on-chip system overcomes the interruption problem of cocrystallization between analytes and matrix molecules due to contaminants from direct injection.It also avoids the complicated off-line pre-treatment of samples.This on-chip separation,purification,and MALDI-TOF MS analysis system is possible to detect target molecules from a complex or an original body fluid solution.%本文通过沉积在多孔硅表面的银纳米粒吸附对氨基苯硫酚和氨基的化学转化得到终端为NiⅡ-Nα,Nα-二(羧甲基)-L-赖氨酸水合物-即Ni Ⅱ-NTA体系的芯片.NiⅡ-NTA修饰的芯片被用于从高浓度的盐和助溶剂的缓冲体系中亲和捕获组氨酸标记的融合蛋白:thioredoxin-urodilatin和SUMO-hu-aprotinin,并进行在线的MALDI-TOF质谱检测,克服了MALDI-TOF质谱中直接点样污染物妨碍样品与基质共结晶的问题,避免了繁琐的离线样品预处理.芯片在线分离、纯化和MALDI-TOF质谱分析体系有望在复杂或原始体液的溶液中分析目标分子.