A molecular scanner to automate proteomic research and to display proteome images
A molecular scanner to automate proteomic research and to display proteome images
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DOI：
10.1021/ac990449e
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发表时间：
1999-11-01
影响因子：
7.4
通讯作者：
Hochstrasser, DF
中科院分区：
文献类型：
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作者：
Binz, PA;Müller, M;Hochstrasser, DF
鉴定和表征生物样品中由基因组表达的所有蛋白质是蛋白质组学中的主要挑战。当今常用的高通量方法将联合收割机二维电泳（2-DE）与肽质量指纹（PMF）分析相结合。虽然自动化通常是可能的，但许多限制仍然对2-DE数据库中的蛋白质鉴定和注释的速率产生不利影响：含有蛋白质的凝胶片的顺序切除过程;酶消化步骤;质谱的解释（鉴定的可靠性）;以及2-DE数据库的手动更新。我们提出了一个高度自动化的方法，生成一个完全注释的2-DE地图。使用一个平行的过程中，所有的蛋白质的2-DE首先同时消化蛋白水解和电转移到聚（偏二氟乙烯）膜上。然后通过MALDI-TOF MS直接扫描膜。在使用PeptIdent软件（http：//www.expasy.ch/tools/peptident.html）从所获得的肽质量指纹自动鉴定蛋白质之后，在线创建完全注释的2-D图谱。它是蛋白质组的多维表示，除了蛋白质鉴定结果外，还包含解释的PMF数据。这种“MS成像”方法代表了临床分子扫描仪发展的重要一步。
Identification and characterization of all proteins expressed by a genome in biological samples represent major challenges in proteomics. Today's commonly used high-throughput approaches combine two-dimensional electrophoresis (2-DE) with peptide mass fingerprinting (PMF) analysis. Although automation is often possible, a number of limitations still adversely affect the rate of protein identification and annotation in 2-DE databases: the sequential excision process of pieces of gel containing protein; the enzymatic digestion step; the interpretation of mass spectra (reliability of identifications); and the manual updating of 2-DE databases. We present a highly automated method that generates a fully annotated 2-DE map. Using a parallel process, all proteins of a 2-DE are first simultaneously digested proteolytically and electrotransferred onto a poly(vinylidene difluoride) membrane. The membrane is then directly scanned by MALDI-TOF MS. After automated protein identification from the obtained peptide mass fingerprints using PeptIdent software (http://www.expasy.ch/tools/peptident.html), a fully annotated 2-D map is created on-line. It is a multidimensional representation of a proteome that contains interpreted PMF data in addition to protein identification results. This "MS-imaging" method represents a major step toward the development of a clinical molecular scanner.