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Sequential Precipitation and Delipidation Enables Efficient Enrichment of Low-Molecular Weight Proteins and Peptides from Human Plasma

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posted on 2020-07-03, 09:29 authored by Na Li, Yue Zhou, Jifeng Wang, Lili Niu, Qing Zhang, Lang Sun, Xiang Ding, Xiaojing Guo, Zhensheng Xie, Nali Zhu, Mengmeng Zhang, Xiulan Chen, Tanxi Cai, Fuquan Yang
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-tert-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.

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