Differential quantitative analysis of MHC ligands by mass spectrometry using stable isotope labeling
Differential quantitative analysis of MHC ligands by mass spectrometry using stable isotope labeling
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DOI：
10.1038/nbt947
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发表时间：
2004-04-01
影响因子：
46.9
通讯作者：
Stevanovic, S
中科院分区：
文献类型：
--
作者：
Lemmel, C;Weik, S;Stevanovic, S
目前，没有方法允许直接和定量比较成对样品中的MHC呈递的肽，例如转染的和未转染的、肿瘤的和正常的或感染的和未感染的组织或细胞系。在这里，我们介绍了两种方法，使用同位素标记的试剂，通过质谱定量的比例，从每个来源的肽。第一种方法涉及乙酰化（1），既快速又简单。然而，通过结合胍基化（2）和烟酰化（3）的第二种方法实现了更高的肽回收率和更精细的灵敏度，因为可以保持肽的电荷状态。使用差异乙酰化，我们确定了一个β连环蛋白衍生肽在实体结肠癌上的人白细胞抗原-A（HLA-A）*6801。将胍基化/烟碱化应用于角蛋白18转染的细胞，并导致肽RLASYLDRV（HLA-A*0201）的表征，其仅存在于转染子上。因此，我们证明了方法，使成对的定量比较，导致过度表达的MHC配体的识别。
Currently, no method allows direct and quantitative comparison of MHC-presented peptides in pairs of samples, such as transfected and untransfected, tumorous and normal or infected and uninfected tissues or cell lines. Here we introduce two approaches that use isotopically labeled reagents to quantify by mass spectrometry the ratio of peptides from each source. The first method involves acetylation(1) and is both fast and simple. However, higher peptide recoveries and a finer sensitivity are achieved by the second method, which combines guanidination(2) and nicotinylation(3), because the charge state of peptides can be maintained. Using differential acetylation, we identified a beta catenin-derived peptide in solid colon carcinoma overpresented on human leucocyte antigen-A (HLA-A)*6801. Guanidination/nicotinylation was applied to keratin 18-transfected cells and resulted in the characterization of the peptide RLASYLDRV (HLA-A*0201), exclusively presented on the transfectant. Thus, we demonstrate methods that enable a pairwise quantitative comparison leading to the identification of overpresented MHC ligands.