Mass spectrometry analysis and quantitation of peptides presented on the MHC II molecules of mouse spleen dendritic cells.
Mass spectrometry analysis and quantitation of peptides presented on the MHC II molecules of mouse spleen dendritic cells.
复制标题
DOI：
10.1021/pr200503g
复制
发表时间：
2011-11-04
影响因子：
4.4
通讯作者：
Steinman, Ralph M.
中科院分区：
文献类型：
--
作者：
Bozzacco, Leonia;Yu, Haiqiang;Zebroski, Henry A., III;Dengjel, Joern;Deng, Haiteng;Mojsov, Svetlana;Steinman, Ralph M.
主要组织相容性复合体II类（MHC II）分子在抗原提呈细胞表面表达，并显示来自自身和非自身抗原的短结合肽片段。这些肽- mhc复合物的功能是在自身抗原的情况下维持免疫耐受，在外来蛋白的情况下启动CD4+ T细胞反应。在这里，我们报告了应用LC-MS/MS分析来鉴定新鲜分离的小鼠脾dc中表达的内源性蛋白衍生的MHC II肽。用Flt3L-B16黑色素瘤细胞处理后，体内细胞数量增加。在一个典型的实验中，从大约5× 108个脾dc开始，我们能够可靠地鉴定出超过100个MHC II肽库，这些肽来自大约55个定位于膜（23%）、细胞内（26%）、内溶酶体（12%）、核（14%）和细胞外（25%）区室的蛋白质。利用合成的与代表性结合MHC II肽序列对应的同位素标记肽，我们用LC-MS相对肽丰度进行定量。在单个实验中，肽的检测浓度范围从2.5 fmol/μL到12 pmol/μL，或从每DC约13拷贝到2×105拷贝。这些多肽在B细胞中发现的数量相似，我们在B细胞中检测到大约80个多肽，这些多肽来自55种蛋白质，均匀分布在与dc相同的细胞区室中。通过表位预测算法预测，在鉴定的MHC II肽序列中发现了约90种不同的结合基序。这些结果为未来定量研究不同免疫条件下产生的dc的MHC II库奠定了基础。
Major histocompatibility complex class II (MHC II) molecules are expressed on the surface of antigen presenting cells and display short bound peptide fragments derived from self and nonself antigens. These peptide-MHC complexes function to maintain immunological tolerance in the case of self antigens and initiate the CD4+ T cell response in the case of foreign proteins. Here we report the application of LC-MS/MS analysis to identify MHC II peptides derived from endogenous proteins expressed in freshly isolated murine splenic DCs. The cell number was enriched in vivo upon treatment with Flt3L-B16 melanoma cells. In a typical experiment, starting with about 5× 108 splenic DCs, we were able to reliably identify a repertoire of over 100 MHC II peptides originating from about 55 proteins localized in membrane (23%), intracellular (26%), endo-lysosomal (12%), nuclear (14%) and extracellular (25%) compartments. Using synthetic isotopically labeled peptides corresponding to the sequences of representative bound MHC II peptides, we quantified by LC-MS relative peptide abundance. In a single experiment, peptides were detected in a wide concentration range spanning from 2.5 fmol/μL to 12 pmol/μL or from approximately 13 copies to 2×105 copies per DC. These peptides were found in similar amounts on B cells where we detected about 80 peptides originating from 55 proteins distributed homogenously within the same cellular compartments as in DCs. About 90 different binding motifs predicted by the epitope prediction algorithm were found within the sequences of the identified MHC II peptides. These results set a foundation for future studies to quantitatively investigate the MHC II repertoire on DCs generated under different immunization conditions.
登录
查看更多内容
DOI：
10.1084/jem.20030107
发表时间：
2003-07-21
期刊：
The Journal of experimental medicine
影响因子：
--
作者：
D'Amico A;Wu L
通讯作者：
Wu L
影响因子：
64.8
作者：
CHICZ, RM;URBAN, RG;STROMINGER, JL
通讯作者：
STROMINGER, JL
影响因子：
64.5
作者：
Foster, LJ;de Hoog, CL;Mann, M
通讯作者：
Mann, M
DOI：
10.1073/pnas.0501190102
发表时间：
2005-05-31
影响因子：
11.1
作者：
Dengjel, J;Schoor, O;Stevanovic, S
通讯作者：
Stevanovic, S
影响因子：
7
作者：
Fissolo, Nicolas;Haag, Sabrina;Weissert, Robert
通讯作者：
Weissert, Robert