Identification and quantitation of MHC class II-bound peptides from mouse spleen dendritic cells by immunoprecipitation and mass spectrometry analysis.
Identification and quantitation of MHC class II-bound peptides from mouse spleen dendritic cells by immunoprecipitation and mass spectrometry analysis.
复制标题
DOI：
10.1007/978-1-62703-589-7_14
复制
发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Yu, Haiqiang
中科院分区：
文献类型：
--
作者：
Bozzacco, Leonia;Yu, Haiqiang
免疫学和免疫疗法的进展需要了解抗原递呈细胞的MHC II类和I类分子上递呈的抗原肽序列。最特化的抗原呈递细胞是树突状细胞（DC）。在过去，可以从小鼠脾脏中分离的DC的数量少，阻止了直接分析DC呈递的MHC II肽库。在这里，我们描述了一个协议，整合免疫学方法（在体内富集小鼠脾DC的Flt 3L治疗和免疫沉淀的MHC II肽复合物），质谱分析和肽合成（LC-MS/MS和定量分析非胰蛋白酶肽），以确定和定量的内源性肽结合到DC上的MHC II分子。所描述的方法产生的定量数据是可重复的和可靠的，足以覆盖广泛的肽拷贝数。我们建议在未来的研究中应用这种方法来定量研究在病毒感染或疫苗开发研究中的不同免疫过程中提出的DC上的MHC II库。
Advances in immunology and immune therapies require knowledge of antigenic peptide sequences that are presented on MHC class II and class I molecules of antigen presenting cells. The most specialized antigen presenting cells are dendritic cells (DCs). In the past, the small number of DCs that can be isolated from mouse spleen prevented direct analysis of the MHC II peptide repertoire presented by DCs. Here we describe a protocol that integrates immunological methods (in vivo enrichment of mouse spleen DCs by Flt3L treatment and immunoprecipation of MHC II-peptide complexes), mass spectrometry analysis and peptide synthesis (LC-MS/MS and quantitation analysis for non tryptic peptides) to identify and quantitate the endogenous peptides that are bound to MHC II molecules on DCs. The described method produces quantitative data that are reproducible and reliable enough to cover a wide range of peptide copy numbers. We propose the application of this method in future studies to quantitatively investigate the MHC II repertoire on DCs presented during viral infections or different immunizations in vaccine development research.