Despite the significance of membrane proteins (MPs) in biological system is indisputable, their specific natures make them notoriously difficult to be analyzed. Particularly, the widely used Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) prefers analyses of hydrophilic cytosolic proteins and has a limited ionization efficiency towards hydrophobic MPs. Herein, a hydrophobic compound (E)-propyl α-Cyano-4-Hydroxyl Cinnamylate (CHCA-C3), a propyl-esterified derivative of α-cyano-4-hydroxycinnamic acid (CHCA), was applied as a contaminant tolerant matrix for high sensitivity MALDI-MS analyses of MPs. With CHCA-C3, the detection limits of hydrophobic peptides were 10- to 100-fold better than those using CHCA. Furthermore, high quality of spectra could be achieved in the presence of high concentration of chaotropes, salts and detergents, as well as human urinary and serum environment. Also, CHCA-C3 could generate uniform sample distribution even in the presence of contaminants. This high contaminant-resistance was revealed to be ascribed to the enhanced hydrophobicity of CHCA-C3 with a lower affinity towards hydrophilic contaminants. The application of CHCA-C3 is further demonstrated by the analysis of trypsin/CNBr digests of bacteriorhodopsin containing seven transmembrane domains (TMDs), which dramatically increased numbers of identified hydrophobic peptides in TMDs and sequence coverage (∼100%). Besides, a combined method by using CHCA-C3 with fluoride solvent and a patterned paraffin plate was established for analysis of integral MPs. We achieved a low detection limit of 10 fmol for integral bacteriorhodopsin, which could not be detected using traditional matrices such as 3,5-dimethoxy-4-hydroxycinamic acid, 2,5-dihydroxyacetophenone even at sample concentration of 10 pmol.

中文翻译：

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用于通过基质辅助激光解吸/电离质谱法轻松检测膜蛋白的高灵敏度和污染物耐受性基质

尽管膜蛋白 (MPs) 在生物系统中的重要性是无可争辩的，但它们的特殊性质使它们很难被分析。特别是，广泛使用的基质辅助激光解吸/电离质谱 (MALDI-MS) 更喜欢分析亲水性胞质蛋白，并且对疏水性 MP 的电离效率有限。在此，疏水化合物 (E)-丙基 α-Cyano-4-Hydroxyl Cinnamylate (CHCA-C3)，α-氰基-4-羟基肉桂酸 (CHCA) 的丙基酯化衍生物，被用作耐污染物基质MP 的高灵敏度 MALDI-MS 分析。使用 CHCA-C3，疏水性肽的检测限比使用 CHCA 的检测限好 10 到 100 倍。此外，在存在高浓度离液剂的情况下可以获得高质量的光谱，盐和清洁剂，以及人体尿液和血清环境。此外，即使存在污染物，CHCA-C3 也能产生均匀的样品分布。这种高抗污染性被证明归因于 CHCA-C3 增强的疏水性，而对亲水性污染物的亲和力较低。CHCA-C3 的应用通过对含有七个跨膜结构域 (TMD) 的细菌视紫红质的胰蛋白酶/CNBr 消化物的分析得到进一步证明，这显着增加了 TMD 中已识别疏水肽的数量和序列覆盖率 (~100%)。此外，建立了CHCA-C3与氟化物溶剂和图案石蜡板的组合方法用于积分MP的分析。我们实现了整合细菌视紫红质的 10 fmol 低检测限，