Muck Alexander, Nesnerová Petra, Pichová Iva, Svatos Ales
Max Planck Institute for Chemical Ecology, Jena, Germany.
Electrophoresis. 2005 Jul;26(14):2835-42. doi: 10.1002/elps.200400014.
A fast protocol for prototyping hydrophobic disposable poly(alkyl methacrylate-co-methyl methacrylate) copolymer sample support arrays for matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) of proteins by atmospheric molding is introduced. The sample support arrays were replicated by molding prepolymer alkyl methacrylate solutions into sandwich molds containing a micromachined silicon master, an aluminum spacer, and glass cover plates, followed by UV-initiated in situ polymerization under atmospheric pressure. The fabrication procedure enables a simultaneous fabrication/modification of single-use polymer arrays by a targeted selection of functional groups of the copolymerized monomers during molding. The one-step modification during the fabrication is demonstrated for enhanced protein adsorption to the modified materials by introduction of hydrophobic butyl-, dodecyl-, and octadecyl groups to the polymer backbone without a need for additional surface coating or derivatization. The MALDI-MS performance of the new polymer chips was tested for spectral measurements of bovine pancreas insulin, horse heart myoglobin, and bovine serum albumin. The protein adsorption to the new hydrophobic copolymer chips was studied for bovine pancreas trypsinogen; the sample desalting parameters, such as time and volume, were optimized for myoglobin as model proteins. A significant signal increase was achieved after efficient desalting of an insect Delta11-desaturase membrane protein fragment from a complex elution buffer (100 mM phosphate, 10 mM tris(hydroxyethyl)aminomethane, 0.5 M NaCl, and 10 mM ethylenediamine tetraacetic acid) on the poly(butyl methacrylate-co-methyl methacrylate) copolymer chip (monomer ratio 8:2 v/v) by simply washing the target zones. The new chips offer reduced sample manipulation and device fabrication times as well as simple operation.
介绍了一种快速协议，用于通过常压成型制备用于蛋白质基质辅助激光解吸/电离质谱（MALDI-MS）的疏水性一次性聚（甲基丙烯酸烷基酯-共-甲基丙烯酸甲酯）共聚物样品支持阵列。通过将预聚物甲基丙烯酸烷基酯溶液模塑到包含微加工硅母版、铝垫片和玻璃盖板的三明治模具中，然后在大气压下进行紫外线引发的原位聚合，来复制样品支持阵列。该制造过程能够通过在成型过程中有针对性地选择共聚单体的官能团，同时制造/修饰一次性聚合物阵列。通过在聚合物主链中引入疏水性的丁基、十二烷基和十八烷基，在制造过程中进行一步修饰，以增强蛋白质对修饰材料的吸附，而无需额外的表面涂层或衍生化。对新型聚合物芯片的MALDI-MS性能进行了测试，用于牛胰腺胰岛素、马心肌红蛋白和牛血清白蛋白的光谱测量。研究了牛胰腺胰蛋白酶原对新型疏水性共聚物芯片的蛋白质吸附；以肌红蛋白为模型蛋白，优化了样品脱盐参数，如时间和体积。通过简单地冲洗目标区域，在聚（甲基丙烯酸丁酯-共-甲基丙烯酸甲酯）共聚物芯片（单体比例8:2 v/v）上对来自复杂洗脱缓冲液（100 mM磷酸盐、10 mM三（羟乙基）氨基甲烷、0.5 M氯化钠和10 mM乙二胺四乙酸）的昆虫Delta11-去饱和酶膜蛋白片段进行有效脱盐后，可以显著提高信号。新型芯片减少了样品处理和设备制造时间，操作也很简单。
