Key Laboratory of Protein and Peptide Pharmaceuticals & Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
J Proteome Res. 2020 Aug 7;19(8):3340-3351. doi: 10.1021/acs.jproteome.0c00232. Epub 2020 Jul 3.
Low-molecular weight proteins and peptides (LMWPs, <30 kDa) in human plasma serve as potential biomarkers or drug targets and are endowed with desirable traits for biological and clinical studies. However, the identification of LMWPs from plasma is retarded by high-abundance proteins, high-molecular weight proteins, and lipids. Here, we present a sequential precipitation and delipidation (SPD) method for the efficient enrichment of LMWPs based on methyl--butyl ether/methanol/water systems. The enriched LMWP sample was analyzed by single-shot liquid chromatography-tandem mass spectrometry employing both HCD and EThcD without tryptic digestion, and 725 peptides were identified on average. The LMWP sample was also digested and analyzed using a bottom-up proteomics pipeline, and 289 proteins were identified, of which 129 (44.6%) proteins were less than 30 kDa and lipoprotein-associated proteins were significantly enriched. Additionally, 25 neuropeptides and 19 long noncoding RNA-encoded polypeptides were identified. Taken together, the SPD method shows good sensitivity and reproducibility when compared with other enrichment methods and has great potential for clinical biomarker discovery and application.
血浆中的低分子量蛋白质和肽（LMWP，<30 kDa）可作为潜在的生物标志物或药物靶点，并且具有用于生物学和临床研究的理想特性。然而，由于高丰度蛋白质、高分子量蛋白质和脂质的存在，血浆中 LMWP 的鉴定受到了阻碍。在这里，我们提出了一种基于甲基叔丁基醚/甲醇/水体系的顺序沉淀和去脂（SPD）方法，用于高效富集 LMWP。富集的 LMWP 样品通过单次液相色谱-串联质谱分析，分别采用 HCD 和 EThcD 进行分析，平均可鉴定出 725 个肽段。LMWP 样品也通过基于自上而下的蛋白质组学分析进行消化和分析，共鉴定出 289 种蛋白质，其中 129 种（44.6%）蛋白质小于 30 kDa，并且脂蛋白相关蛋白得到了显著富集。此外，还鉴定出 25 种神经肽和 19 种长非编码 RNA 编码多肽。总的来说，与其他富集方法相比，SPD 方法具有良好的灵敏度和重现性，在临床生物标志物的发现和应用方面具有巨大的潜力。
