Harkins James B, Katz Benjamin B, Pastor Salvador J, Osucha Peter, Hafeman Dean G, Witkowski Charles E, Norris Jeremy L
Protein Discovery, Incorporated, 418 South Gay Street, Suite 203, Knoxville, Tennessee 37902, USA.
Anal Chem. 2008 Apr 15;80(8):2734-43. doi: 10.1021/ac702214n. Epub 2008 Mar 11.
The preparation of complex biological samples for high-throughput mass spectrometric analyses remains a significant bottleneck, limiting advancement of the capabilities of mass spectrometry (MS) and ultimately limiting development of novel clinical assays. The removal of interfering species (e.g., salts, detergents, and buffers), concentration of dilute analytes, and the reduction of sample complexity are required in order to maximize the quality of resultant MS data. This study describes a novel sample preparation method that makes use of electrophoresis to prepare complex biological samples for high-throughput MS analysis. The method provides for integration of key sample preparation steps, including depletion, fractionation, desalting, and concentration. The prepared samples are captured onto a monolithic reversed-phase capture target that can be analyzed directly by a mass spectrometer. Up to 96 individual samples are simultaneously prepared for MS analysis in under 1 h. For standard proteins added to serum, this method provides femtomole level sensitivity and reproducible label-free detection (coefficient of variation <30%). This study demonstrates that this electrophoretic sample preparation system permits high-throughput sample preparation for mass spectrometric analysis of complex biological samples, such as serum, plasma, and tissue extracts.
为高通量质谱分析制备复杂生物样品仍然是一个重大瓶颈，限制了质谱（MS）能力的提升，最终也限制了新型临床检测方法的发展。为了最大化所得质谱数据的质量，需要去除干扰物质（如盐、去污剂和缓冲液）、浓缩稀释的分析物并降低样品复杂性。本研究描述了一种新颖的样品制备方法，该方法利用电泳为高通量质谱分析制备复杂生物样品。该方法实现了关键样品制备步骤的整合，包括去除、分级分离、脱盐和浓缩。制备好的样品被捕获到整体反相捕获靶上，可直接用质谱仪进行分析。在不到1小时的时间内可同时为多达96个独立样品制备用于质谱分析的样品。对于添加到血清中的标准蛋白质，该方法提供飞摩尔级别的灵敏度和可重复的无标记检测（变异系数<30%）。本研究表明，这种电泳样品制备系统允许对复杂生物样品（如血清、血浆和组织提取物）进行高通量样品制备以用于质谱分析。
