使用自上而下质谱法分析低分子量血浆蛋白质组

Low-Molecular-Weight Plasma Proteome Analysis Using Top-Down Mass Spectrometry.

作者信息

Cheon Dong Huey, Yang Eun Gyeong, Lee Cheolju, Lee Ji Eun

机构信息

Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, Republic of Korea.

Interdisciplinary program of Integrated Biotechnology, Sogang University, Seoul, Republic of Korea.

出版信息

Methods Mol Biol. 2017;1619:103-117. doi: 10.1007/978-1-4939-7057-5_8.

DOI:10.1007/978-1-4939-7057-5_8

PMID:28674880

Abstract

While human plasma has a wealth of diagnostic information regarding the state of the human body in heath and disease, low molecular weight (LMW) proteome (<30 kDa) has been shown to contain a rich source of diagnostic biomarkers. Here we describe a protocol for top-down proteomic analysis to identify and characterize the LMW proteoforms present in four types of human plasma samples without immunoaffinity depletion and with depletion of the top two, six, and seven high-abundance proteins. Each type of plasma sample was first fractionated based on molecular weight using gel-eluted liquid fraction entrapment electrophoresis (GELFrEE). Then, the GELFrEE fractions containing up to 30 kDa were subjected to nanocapillary-LC-MS/MS, and the high-resolution MS and MS/MS data were processed using ProSightPC software. As a result, a total of 442 LMW proteins and cleaved products, including those with posttranslational modifications (PTMs) and single amino acid variations (SAAVs), were identified with a threshold E-value of 1 × 10 from the four types of plasma samples.

摘要

虽然人类血浆拥有大量关于人体健康和疾病状态的诊断信息，但低分子量（LMW）蛋白质组（<30 kDa）已被证明含有丰富的诊断生物标志物来源。在此，我们描述了一种自上而下的蛋白质组学分析方案，用于鉴定和表征四种类型的人类血浆样本中存在的LMW蛋白质变体，且不进行免疫亲和去除以及去除前两种、六种和七种高丰度蛋白质。每种类型的血浆样本首先使用凝胶洗脱液相分数截留电泳（GELFrEE）基于分子量进行分级分离。然后，将含有高达30 kDa的GELFrEE级分进行纳升毛细管液相色谱-串联质谱分析（nanocapillary-LC-MS/MS），并使用ProSightPC软件处理高分辨率质谱和串联质谱数据。结果，从四种类型的血浆样本中，以1×10的阈值E值鉴定出总共442种LMW蛋白质和裂解产物，包括那些具有翻译后修饰（PTM）和单氨基酸变异（SAAV）的产物。