Mass spectrometry after capture and small-volume elution of analyte from a surface plasmon resonance biosensor.
Mass spectrometry after capture and small-volume elution of analyte from a surface plasmon resonance biosensor.
复制标题
从表面等离子体共振生物传感器捕获和小体积洗脱分析物后进行质谱分析。
DOI：
10.1021/ac015642z
复制
发表时间：
2002
影响因子：
7.4
通讯作者：
Yergey,AlfredL
中科院分区：
文献类型：
--
作者：
Gilligan,JohnJ;Schuck,Peter;Yergey,AlfredL
在生物传感器表面特异性捕获后用质谱仪鉴定蛋白质的结合伙伴是蛋白质组学研究以及蛋白质−和蛋白质相互作用的鉴定和表征的一种很有前途的工具。以前的方法包括在基质辅助激光解吸电离飞行时间质谱仪(MALDI-TOFMS)上直接电离生物传感器芯片上的分析物，然后在芯片上消化，然后洗脱，碎片的色谱浓度和电喷雾质谱仪。本文利用最近报道的具有振荡流动的小体积微流控样品处理技术(Abrantes et al.Anal.化学2001，73,2828−2835)，分析物被从传感器表面洗脱到促进从捕获表面解离并输送到质谱仪的小体积缓冲液中。传感器表面与样品的孵化和分析物的回收都可以用几个微升来实现，并进行到达到稳定状态。由于该过程对传感器表面是非破坏性的，因此多次捕获和洗脱允许分析物转移和浓缩到洗脱缓冲液中。洗脱后的分析物可以直接用MALDI-TOFMS进行研究，也可以用蛋白水解法进行蛋白质鉴定。转移到洗脱缓冲液中，MALDI-TOFMS从5μL的起始样品中进行检测，样品中含有50fmol的分析物。给出了从胞浆蛋白的复杂混合物中具体检测和回收蛋白质的实例。
The identification of binding partners of proteins by mass spectrometry following specific capture on a biosensor surface is a promising tool for proteomics research and the identification and characterization of protein−protein interactions. Previous approaches include the direct ionization of analyte from the biosensor chip on a matrix assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOFMS) apparatus and the on-chip digestion followed by elution, chromatographic concentration of the fragments, and electrospray mass spectrometry. In the present paper, using the small-volume microfluidic sample manipulation technique with oscillatory flow reported recently (Abrantes et al.Anal. Chem.2001, 73, 2828−2835), analyte is shown to be eluted from the sensor surface into a small volume of buffer that promotes dissociation from the capture surface and delivery to the mass spectrometer. Both the incubation of the sensor surface with the sample and the recovery of analyte can be achieved with a few microliters and conducted until steady-state is attained. Because the procedure is nondestructive for the sensor surface, multiple cycles of capture and elution allow the transfer and concentration of analyte into the elution buffer. The eluted analyte can be studied directly by MALDI-TOFMS, or subjected to proteolytic digestion for protein identification. Transfer into the elution buffer and MALDI-TOFMS detection was achieved from 5 μL of starting samples containing <50 fmol of analyte. Examples are presented for the specific detection and recovery of a protein from a complex mixture of cytosolic proteins.