Sato Toshitaka, Ishihama Yasushi, Oda Yoshiya
Eisai Co., Ltd., Tsukuba, Ibaraki, Japan.
Methods Mol Biol. 2007;359:53-70. doi: 10.1007/978-1-59745-255-7_4.
We describe a new method for quantitative tissue proteomics using culture-derived isotope tags (CDIT), which are cells grown in stable isotope-enriched medium and added to each tissue sample to provide internal standards. After protein identification by mass spectrometry (MS), each peak derived from tissue protein is quantified relative to the corresponding CDIT peak. The amounts of each peak in different tissue samples can be compared relative to CDIT. Even if the corresponding peak from CDIT can not be detected, a peak with a similar scan number, but different sequence on liquid chromatography (LC)-MS, can be used to obtain semiquantitative values. Absolute quantification is possible by determining the protein amount in CDIT in advance using unlabeled synthetic peptides; this is less costly than other methods, such as AQUA. For identification of specific components in a protein complex, target proteins are enriched or isolated by affinity techniques using bait-conjugated matrix, but many nonspecific binders are often found. Stable isotope labeling strategies have proven particularly advantageous for the discrimination of proteins specifically associated with the target population from nonspecifically, copurified contaminants. We also describe a protocol for efficient in-gel digestion and high-performance nano-LC column preparation, which makes it possible to quantify larger numbers of proteins.
我们描述了一种使用培养衍生同位素标签（CDIT）进行定量组织蛋白质组学的新方法，CDIT是在富含稳定同位素的培养基中生长的细胞，并添加到每个组织样本中以提供内标。通过质谱（MS）鉴定蛋白质后，将来自组织蛋白质的每个峰相对于相应的CDIT峰进行定量。不同组织样本中每个峰的量可以相对于CDIT进行比较。即使无法检测到来自CDIT的相应峰，具有相似扫描编号但在液相色谱（LC）-MS上序列不同的峰也可用于获得半定量值。通过预先使用未标记的合成肽确定CDIT中的蛋白质量，可以进行绝对定量；这比其他方法（如AQUA）成本更低。为了鉴定蛋白质复合物中的特定成分，使用与诱饵偶联的基质通过亲和技术富集或分离目标蛋白，但经常会发现许多非特异性结合物。稳定同位素标记策略已被证明对于从非特异性共纯化污染物中区分与目标群体特异性相关的蛋白质特别有利。我们还描述了一种高效的胶内消化和高性能纳米LC柱制备方案，这使得对更多数量的蛋白质进行定量成为可能。
