A high sensitive and contaminant tolerant matrix for facile detection of membrane proteins by matrix-assisted laser desorption/ionization mass spectrometry
A high sensitive and contaminant tolerant matrix for facile detection of membrane proteins by matrix-assisted laser desorption/ionization mass spectrometry
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一种高灵敏度和耐污染基质，可通过基质辅助激光解吸/电离质谱法轻松检测膜蛋白
DOI：
10.1016/j.aca.2017.11.018
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发表时间：
2018
影响因子：
6.2
通讯作者：
Guo Xinhua
中科院分区：
文献类型：
--
作者：
Wang Sheng;Xiao Chunsheng;Jiang Liyan;Ling Ling;Chen Xuesi;Guo Xinhua
尽管膜蛋白在生物系统中的重要性是毋庸置疑的，但其特殊的性质使其难以分析。特别地，广泛使用的基质辅助激光解吸/电离质谱（MALDI-MS）优选分析亲水性胞质蛋白，并且对疏水性MP具有有限的电离效率。本文以α-氰基-4-羟基肉桂酸（CHCA）的丙酯化衍生物（E）-α-氰基-4-羟基肉桂酸丙酯（CHCA-C3）为基质，对MP进行了高灵敏度MALDI-MS分析。使用CHCA-C3，疏水肽的检测限比使用CHCA的检测限高10至100倍。此外，在高浓度离液剂、盐和去污剂以及人尿液和血清环境的存在下，可以实现高质量的光谱。此外，CHCA-C3可以产生均匀的样品分布，即使在污染物的存在下。这种高耐污染性被揭示归因于CHCA-C3的增强的疏水性，对亲水性污染物具有较低的亲和力。通过对含有7个跨膜结构域（TMDs）的细菌视紫红质的胰蛋白酶/CNBr酶切分析，CHCA-C3的应用得到进一步证明，这显著增加了TMDs中鉴定的疏水肽的数量和序列覆盖率（约100%）。此外，还建立了CHCA-C3-氟溶剂-图案化石蜡板联用分析整体MPs的方法。我们实现了10 fmol的低检测限的整体细菌视紫红质，这不能检测到使用传统的矩阵，如3，5-二甲氧基-4-羟基肉桂酸，2，5-二羟基苯乙酮，甚至在样品浓度为10 pmol。
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.