Feist Peter, Hummon Amanda B
Department of Chemistry and Biochemistry, Integrated Biomedical Sciences Program, and the Harper Cancer Research Institute, 251 Nieuwland Science Hall, University of Notre Dame, Notre Dame, IN 46556, USA.
Int J Mol Sci. 2015 Feb 5;16(2):3537-63. doi: 10.3390/ijms16023537.
Proteins regulate many cellular functions and analyzing the presence and abundance of proteins in biological samples are central focuses in proteomics. The discovery and validation of biomarkers, pathways, and drug targets for various diseases can be accomplished using mass spectrometry-based proteomics. However, with mass-limited samples like tumor biopsies, it can be challenging to obtain sufficient amounts of proteins to generate high-quality mass spectrometric data. Techniques developed for macroscale quantities recover sufficient amounts of protein from milligram quantities of starting material, but sample losses become crippling with these techniques when only microgram amounts of material are available. To combat this challenge, proteomicists have developed micro-scale techniques that are compatible with decreased sample size (100 μg or lower) and still enable excellent proteome coverage. Extraction, contaminant removal, protein quantitation, and sample handling techniques for the microgram protein range are reviewed here, with an emphasis on liquid chromatography and bottom-up mass spectrometry-compatible techniques. Also, a range of biological specimens, including mammalian tissues and model cell culture systems, are discussed.
蛋白质调节许多细胞功能，分析生物样品中蛋白质的存在和丰度是蛋白质组学的核心重点。使用基于质谱的蛋白质组学可以实现各种疾病的生物标志物、信号通路和药物靶点的发现与验证。然而，对于像肿瘤活检这样的微量样本，要获得足够量的蛋白质以生成高质量的质谱数据可能具有挑战性。为宏观量样本开发的技术能够从毫克级的起始材料中回收足够量的蛋白质，但当只有微克级的材料可用时，这些技术的样本损失就会变得很严重。为应对这一挑战，蛋白质组学研究人员开发了与减少的样本量（100μg或更低）兼容且仍能实现出色蛋白质组覆盖的微量技术。本文综述了微克级蛋白质范围内的提取、污染物去除、蛋白质定量和样本处理技术，重点介绍了液相色谱和自下而上质谱兼容技术。此外，还讨论了一系列生物标本，包括哺乳动物组织和模型细胞培养系统。
