Kartanas Tadas, Levin Aviad, Toprakcioglu Zenon, Scheidt Tom, Hakala Tuuli A, Charmet Jerome, Knowles Tuomas P J
Centre for Misfolding Diseases, Yusuf Hamied Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K.
WMG, University of Warwick, Coventry CV4 7AL, U.K.
Anal Chem. 2021 Feb 9;93(5):2848-2853. doi: 10.1021/acs.analchem.0c04149. Epub 2021 Jan 28.
The detection and analysis of proteins in a label-free manner under native solution conditions is an increasingly important objective in analytical bioscience platform development. Common approaches to detect native proteins in solution often require specific labels to enhance sensitivity. Dry mass sensing approaches, by contrast, using mechanical resonators, can operate in a label-free manner and offer attractive sensitivity. However, such approaches typically suffer from a lack of analyte selectivity as the interface between standard protein separation techniques and micro-resonator platforms is often constrained by qualitative mechanical sensor performance in the liquid phase. Here, we describe a strategy that overcomes this limitation by coupling liquid chromatography with a quartz crystal microbalance (QCM) platform by using a microfluidic spray dryer. We explore a strategy which allows first to separate a protein mixture in a physiological buffer solution using size exclusion chromatography, permitting specific protein fractions to be selected, desalted, and subsequently spray-dried onto the QCM for absolute mass analysis. By establishing a continuous flow interface between the chromatography column and the spray device via a flow splitter, simultaneous protein mass detection and sample fractionation is achieved, with sensitivity down to a 100 μg/mL limit of detection. This approach for quantitative label-free protein mixture analysis offers the potential for detection of protein species under physiological conditions.
在天然溶液条件下以无标记方式检测和分析蛋白质是分析生物科学平台开发中一个日益重要的目标。溶液中检测天然蛋白质的常用方法通常需要特定标记来提高灵敏度。相比之下，使用机械谐振器的干质量传感方法可以以无标记方式运行并具有吸引人的灵敏度。然而，由于标准蛋白质分离技术与微谐振器平台之间的界面通常受到液相中定性机械传感器性能的限制，此类方法通常存在缺乏分析物选择性的问题。在此，我们描述了一种策略，该策略通过使用微流体喷雾干燥器将液相色谱与石英晶体微天平（QCM）平台相结合来克服这一限制。我们探索了一种策略，该策略首先允许使用尺寸排阻色谱法在生理缓冲溶液中分离蛋白质混合物，从而可以选择特定的蛋白质组分、脱盐，随后喷雾干燥到QCM上进行绝对质量分析。通过使用分流器在色谱柱和喷雾装置之间建立连续流动界面，实现了蛋白质质量的同时检测和样品分级分离，检测限低至100μg/mL。这种用于定量无标记蛋白质混合物分析的方法为在生理条件下检测蛋白质种类提供了可能性。
