Comprehensive proteomic mass spectrometric characterization of human cannabinoid CB2 receptor
Comprehensive proteomic mass spectrometric characterization of human cannabinoid CB2 receptor
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DOI：
10.1021/pr060671h
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发表时间：
2007-01-01
影响因子：
4.4
通讯作者：
Makriyannis, Alexandros
中科院分区：
文献类型：
--
作者：
Zvonok, Nikolai;Yaddanapudi, Suma;Makriyannis, Alexandros
CB1 和 CB2 大麻素受体属于 GPCR 超家族，与多种生理和病理生理过程相关。这两种受体以及几种处于不同开发阶段的先导化合物都是药物发现的潜在有用靶标。因此，充分阐明这些膜相关蛋白的结构特征对于设计更具选择性的新型治疗药物分子非常有价值。作为获得药物-受体复合物结构特征信息的第一步，我们描述了重组人大麻素 CB2 受体的完整质谱 (MS) 分析。首次对视紫红质以外的 GPCR 蛋白进行完整的蛋白质组学表征，是通过结合多种 LC/MS 方法（涉及纳米毛细管液相色谱）以及四极杆线性离子阱或线性离子阱 FTICR 质谱仪来完成的。带有掺入 N 端 FLAG 和 C 端 HIS6 表位标签的 CB2 受体在杆状病毒细胞中功能性表达，并使用抗 FLAG M2 亲和层析的一步纯化。为了克服凝胶内消化所涉及的困难，由于这种膜相关蛋白的高度疏水性，我们在低浓度 CYMAL5 存在的情况下对纯化和脱盐的样品进行了溶液内胰蛋白酶和糜蛋白酶消化。随后使用以正模式运行的纳喷雾 ESI 源进行 nanoLC 肽分离和分析。基于使用高质量精确 LTQ-FT 和 4000 Q-Trap 质谱仪获得的重叠序列数据，可以自信地报告结果。两种仪器都给出了非常相似的已识别肽模式，完全覆盖了所有跨膜螺旋，从而完整地表征了大麻素 CB2 受体。大麻素 CB2 受体中所有氨基酸残基的质谱鉴定是我们实验室开发的“基于配体的结构生物学”方法的关键一步，该方法使用各种共价大麻能配体来表征 GPCR 中的配体结合位点。
The CB1 and CB2 cannabinoid receptors belong to the GPCR superfamily and are associated with a variety of physiological and pathophysiological processes. Both receptors, with several lead compounds at different phases of development, are potentially useful targets for drug discovery. For this reason, fully elucidating the structural features of these membrane-associated proteins would be extremely valuable in designing more selective, novel therapeutic drug molecules. As a first step toward obtaining information on the structural features of the drug-receptor complex, we describe the full mass spectrometric ( MS) analysis of the recombinant human cannabinoid CB2 receptor. This first complete proteomic characterization of a GPCR protein beyond rhodopsin was accomplished by a combination of several LC/MS approaches involving nanocapillary liquid chromatography, coupled with either a quadrupole-linear ion trap or linear ion trap-FTICR mass spectrometer. The CB2 receptor, with incorporated N-terminal FLAG and C-terminal HIS6 epitope tags, was functionally expressed in baculovirus cells and purified using a single step of anti-FLAG M2 affinity chromatography. To overcome the difficulties involved with in-gel digestion, due to the highly hydrophobic nature of this membrane-associated protein, we conducted in-solution trypsin and chymotrypsin digestions of purified and desalted samples in the presence of a low concentration of CYMAL5. This was followed by nanoLC peptide separation and analysis using a nanospray ESI source operated in the positive mode. The results can be reported confidently, based on the overlapping sequence data obtained using the highly mass accurate LTQ-FT and the 4000 Q-Trap mass spectrometers. Both instruments gave very similar patterns of identified peptides, with full coverage of all transmembrane helices, resulting in the complete characterization of the cannabinoid CB2 receptor. Mass spectrometric identification of all amino acid residues in the cannabinoid CB2 receptor is a key step toward the "Ligand Based Structural Biology" approach developed in our laboratory for characterizing ligand binding sites in GPCRs using a variety of covalent cannabinergic ligands.