Bennike Tue Bjerg, Steen Hanno
Department of Pathology, Boston Children's Hospital and Harvard Medical School, Boston, MA, 02115, USA.
Methods Mol Biol. 2017;1619:395-402. doi: 10.1007/978-1-4939-7057-5_27.
Meaningful proteomic-based biomarker discovery projects using primary human-derived specimens require the analysis of hundreds of samples in order to address the issue of interpersonal variability. Thus, robust high-throughput methods for the digestion of plasma samples are a prerequisite for such large clinical proteomic studies with hundreds of samples. Commonly used sample preparation methods are often difficult to parallelize and/or automate. Herein we describe a method for parallel 96-well plate-based sample preparation. Protein digestion is performed in 96-well polyvinylidene fluoride (PVDF) membrane plates and the subsequent purification in 96-well reversed phase C18 purification plates, enabling the usage of multichannel pipettes in all steps. The protocol can be applied using neat or depleted plasma/serum samples, but has also proven effective with other sample types.
使用源自人类原代样本进行基于蛋白质组学的有意义的生物标志物发现项目，需要分析数百个样本，以解决个体间变异性问题。因此，用于血浆样本消化的强大高通量方法是此类涉及数百个样本的大型临床蛋白质组学研究的先决条件。常用的样本制备方法通常难以并行化和/或自动化。在此，我们描述一种基于96孔板的并行样本制备方法。蛋白质消化在96孔聚偏二氟乙烯（PVDF）膜板中进行，随后在96孔反相C18纯化板中进行纯化，从而在所有步骤中都能使用多通道移液器。该方案可应用于纯血浆/血清样本或去除特定成分的血浆/血清样本，但也已证明对其他样本类型有效。
