Proteolytic 18O labeling for comparative proteomics:: Model studies with two serotypes of adenovirus
Proteolytic 18O labeling for comparative proteomics:: Model studies with two serotypes of adenovirus
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DOI：
10.1021/ac001404c
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发表时间：
2001-07-01
影响因子：
7.4
通讯作者：
Fenselau, C
中科院分区：
文献类型：
--
作者：
Yao, XD;Freas, A;Fenselau, C
引入了一种用于蛋白水解稳定同位素标记的新方法，以提供来自两个整个蛋白质组库或其亚组分的单个蛋白质之间的定量和并发比较。在第一个池中所有蛋白质的蛋白水解裂解过程中，两个 O-18 原子普遍并入所有胰蛋白酶肽的羧基末端。第二池中的蛋白质与含有两个 O-16 原子的所得肽的羧基末端类似地被切割（即，无标记）。将两种肽混合物合并进行分级和分离，并通过高分辨率质谱法测量每个肽对的质量和同位素比率（相差 4 Da）。短序列和/或精确质量测量与蛋白质组学软件工具相结合，使肽能够与它们所衍生的前体蛋白相关联。配对肽的相对信号强度量化了要比较的蛋白质组库中其前体蛋白的表达水平，使用论文中描述的方程，单个（未配对）肽的观察主要被解释为来自各自蛋白质组库的蛋白质的差异修饰或序列变异，该方法在此评估腺病毒两种血清型（Ad5和Ad2）的病毒体蛋白的比较，利用有关蛋白质序列和浓度的现有信息。一般来说，蛋白水解 O-18 标记为具有定量能力的蛋白质组学研究提供了鸟枪法，并被提议作为非常复杂的蛋白质混合物的比较蛋白质组学研究的有用工具。
A new method for proteolytic stable isotope labeling is introduced to provide quantitative and concurrent comparisons between individual proteins from two entire proteome pools or their subfractions. Two O-18 atoms are incorporated universally into the carboxyl termini of all tryptic peptides during the proteolytic cleavage of all proteins in the first pool. Proteins in the second pool are cleaved analogously with the carboxyl termini of the resulting peptides containing two O-16 atoms (i.e., no labeling). The two peptide mixtures are pooled for fractionation and separation, and the masses and isotope ratios of each peptide pair (differing by 4 Da) are measured by high-resolution mass spectrometry, Short sequences and/or accurate mass measurements combined with proteomics software tools allow the peptides to be related to the precursor proteins from which they are derived. Relative signal intensities of paired peptides quantify the expression levels of their precursor proteins from proteome pools to be compared, using an equation described in the paper, Observation of individual (unpaired) peptides is mainly interpreted as differential modification or sequence variation for the protein from the respective proteome pool, The method is evaluated here in a comparison of virion proteins for two serotypes (Ad5 and Ad2) of adenovirus, taking advantage of information already available about protein sequences and concentrations. In general, proteolytic O-18 labeling enables a shotgun approach for proteomic studies with quantitation capability and is proposed as a useful tool for comparative proteomic studies of very complex protein mixtures.