Bantscheff Marcus, Dümpelfeld Birgit, Kuster Bernhard
Cellzome AG, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
Rapid Commun Mass Spectrom. 2004;18(8):869-76. doi: 10.1002/rcm.1418.
Stable isotope labeling (SIL) has emerged as a powerful tool to measure the relative quantitative differences between samples in many differential display-type proteomic applications. However, current SIL procedures tend to suffer from the fact that one needs to decide very early in a biochemical strategy whether or not a sample will be subjected to relative quantification. Typically, the entire strategy has to be adapted to the needs of the particular quantification method chosen which might limit the range of biochemical experiments amenable to quantification. Metabolic labeling approaches, albeit very sensitive, can only be applied to studies using appropriate cell culture systems which might not necessarily be compatible with the biological system under investigation. Chemical labeling of complex protein mixtures by, e.g., isotope-coded affinity tags (ICAT), can offer great simplification of protein mixtures but is restricted by the accessibility of the often few suitable peptides (i.e. cysteine containing peptides) for both protein identification and quantification. Here, we describe a post-digest (18)O-labeling method that can circumvent some of the above limitations by separating protein identification from quantification. An aliquot of all samples in a set can be used for rapid protein ID using, e.g., matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). In a second step, relative quantification is performed using trypsin-catalyzed (18)O incorporation into all tryptic peptides. This two-stage procedure introduces significant experimental flexibility because it enables postponement of the decision about which pairs of samples from a given set of experiments are to be compared until after the protein ID stage. In-gel digested protein quantities between 50 fmol and 15 pmol are amenable to this new method, with a dynamic range of 1:10 within one sample. Accuracy for measured relative abundances is similar to those reported for other SIL strategies (errors typically <20%), and the method is applicable to protein samples from all kinds of tissue or cell culture. This paper presents quantification data for a set of standard proteins, as well as a study of differential complex formation around the NFkappaB transcription factor p65 following stimulation with TNF-alpha.
在许多差异显示型蛋白质组学应用中，稳定同位素标记（SIL）已成为测量样品间相对定量差异的强大工具。然而，目前的SIL方法往往存在这样的问题：在生化策略的早期阶段，就需要决定一个样品是否将进行相对定量分析。通常情况下，整个策略必须适应所选特定定量方法的需求，这可能会限制适用于定量分析的生化实验范围。代谢标记方法虽然非常灵敏，但仅适用于使用合适细胞培养系统的研究，而这些系统不一定与所研究的生物系统兼容。例如，通过同位素编码亲和标签（ICAT）对复杂蛋白质混合物进行化学标记，虽然可以极大地简化蛋白质混合物，但受到常用于蛋白质鉴定和定量分析的合适肽段（即含半胱氨酸的肽段）数量有限的限制。在此，我们描述了一种消化后（18）O标记方法，该方法通过将蛋白质鉴定与定量分析分离，可规避上述一些限制。一组样品中的等分试样可用于通过例如基质辅助激光解吸/电离飞行时间质谱（MALDI-TOFMS）进行快速蛋白质鉴定。第二步，使用胰蛋白酶催化（18）O掺入所有胰蛋白酶肽段来进行相对定量分析。这种两阶段程序引入了显著的实验灵活性，因为它能够将关于给定实验集中哪些样品对进行比较的决定推迟到蛋白质鉴定阶段之后。凝胶内消化的蛋白质量在50 fmol至15 pmol之间适用于这种新方法，一个样品内的动态范围为1:10。所测相对丰度的准确度与其他SIL策略报告的类似（误差通常<20%），并且该方法适用于来自各种组织或细胞培养的蛋白质样品。本文展示了一组标准蛋白质的定量数据，以及一项关于用TNF-α刺激后围绕NFκB转录因子p65的差异复合物形成的研究。
