National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, Maryland 20850, United States.
Division of Nephrology, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201, United States.
Anal Chem. 2021 Jun 22;93(24):8423-8431. doi: 10.1021/acs.analchem.1c00265. Epub 2021 Jun 10.
Major advances have been made to improve the sensitivity of mass analyzers, spectral quality, and speed of data processing enabling more comprehensive proteome discovery and quantitation. While focus has recently begun shifting toward robust proteomics sample preparation efforts, a high-throughput proteomics sample preparation is still lacking. We report the development of a highly automated universal 384-well plate sample preparation platform with high reproducibility and adaptability for extraction of proteins from cells within a culture plate. Digestion efficiency was excellent in comparison to a commercial digest peptide standard with minimal sample loss while improving sample preparation throughput by 20- to 40-fold (the entire process from plated cells to clean peptides is complete in ∼300 min). Analysis of six human cell types, including two primary cell samples, identified and quantified ∼4,000 proteins for each sample in a single high-performance liquid chromatography (HPLC)-tandem mass spectrometry injection with only 100-10K cells, thus demonstrating universality of the platform. The selected protein was further quantified using a developed HPLC-multiple reaction monitoring method for HeLa digests with two heavy labeled internal standard peptides spiked in. Excellent linearity was achieved across different cell numbers indicating a potential for target protein quantitation in clinical research.
在提高质谱仪的灵敏度、光谱质量和数据处理速度方面已经取得了重大进展，从而能够更全面地发现和定量蛋白质组。虽然最近的研究重点开始转向稳健的蛋白质组学样品制备工作，但高通量蛋白质组学样品制备仍然缺乏。我们报告了一种高度自动化的通用 384 孔板样品制备平台的开发，该平台具有高重现性和适应性，可从培养板中的细胞中提取蛋白质。与商业消化肽标准品相比，该平台的消化效率非常出色，几乎没有样品损失，同时将样品制备通量提高了 20-40 倍（从 plated 细胞到清洁肽的整个过程在约 300 分钟内完成）。对六种人类细胞类型（包括两个原代细胞样本）的分析表明，每个样本的鉴定和定量蛋白质数量约为 4000 种，单次高效液相色谱（HPLC）-串联质谱注射只需 100-10K 个细胞，从而证明了该平台的通用性。使用两种重标记内标肽掺入的 HeLa 消化物开发的 HPLC-多重反应监测方法进一步对选定的蛋白质进行定量，结果表明该方法具有良好的线性度，适用于不同数量的细胞，有望用于临床研究中的目标蛋白定量。
