Differential expression analysis of Escherichia coli proteins using a novel software for relative quantitation of LC-MS/MS data
Differential expression analysis of Escherichia coli proteins using a novel software for relative quantitation of LC-MS/MS data
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DOI：
10.1002/pmic.200500921
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发表时间：
2006-08-01
期刊：
影响因子：
3.4
通讯作者：
Flensburg, John
中科院分区：
文献类型：
--
作者：
Johansson, Carolina;Samskog, Jenny;Flensburg, John
研究代表不同生物条件的细胞样品之间蛋白质水平的变化是理解细胞功能的关键。有两种主要的方法可用，既可以对显著变化的蛋白质进行全局扫描，也可以对感兴趣的蛋白质进行靶向分析。一种方法是基于二维凝胶电泳和标记蛋白的图像分析。另一种方法是基于LC-MS/MS分析未标记的肽或来自同位素标记的蛋白质或肽的肽。在本研究中，采用非标记方法，使用一种新的软件DeCydei (TM) S差分分析软件（DeCyder MS），用于LC-MS/MS数据中未标记肽的自动检测和相对定量。用胰蛋白酶消化表达不同水平二氢叶酸还原酶和整合子整合酶的大肠杆菌菌株的总蛋白提取物，并使用纳米级液相色谱系统etan (TM) MDLC进行分析，该系统与配备纳米喷雾接口的LTQ （TM）线性离子阱质谱仪在线连接。获取的NIS数据进行DeCyder NIS分析，其中来自单个LC-MS/MS分析的肽模式的二维表示进行匹配和比较。这种对大肠杆菌蛋白质组进行无标记定量分析的方法得到的相对蛋白质丰度与测量蛋白质水平的传统方法得到的结果非常一致。
The study of changes in protein levels between samples derived from cells representing different biological conditions is a key to the understanding of cellular function. There are two main methods available that allow both for global scanning for significantly varying proteins and targeted profiling of proteins of interest. One method is based on 2-D gel electrophoresis and image analysis of labelled proteins. The other method is based on LC-MS/MS analysis of either unlabelled peptides or peptides derived from isotopically labelled proteins or peptides. In this study, the non-labelling approach was used involving a new software, DeCydei (TM) S Differential Analysis Software (DeCyder MS) intended for automated detection and relative quantitation of unlabelled peptides in LC-MS/MS data. Total protein extracts of E. coli strains expressing varying levels of dihydrofolate reductase and integron integrase were digested with trypsin and analyzed using a nanoscale liquid chromatography system, Ettan (TM) MDLC, online connected to an LTQ (TM) linear ion-trap mass spectrometer fitted with a nanospray interface. Acquired NIS data were subjected to DeCyder NIS analysis where 2-D representations of the peptide patterns from individual LC-MS/MS analyses were matched and compared. This approach to unlabelled quantitative analysis of the E. coli proteome resulted in relative protein abundances that were in good agreement with results obtained from traditional methods for measuring protein levels.