Control of Selectivity via Nanochemistry: Monolithic Capillary Column Containing Hydroxyapatite Nanoparticles for Separation of Proteins and Enrichment of Phosphopeptides
Control of Selectivity via Nanochemistry: Monolithic Capillary Column Containing Hydroxyapatite Nanoparticles for Separation of Proteins and Enrichment of Phosphopeptides
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DOI：
10.1021/ac1018815
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发表时间：
2010-10-01
影响因子：
7.4
通讯作者：
Svec, Frantisek
中科院分区：
文献类型：
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作者：
Krenkova, Jana;Lacher, Nathan A.;Svec, Frantisek
新型整体式毛细管柱嵌入了商业羟基磷灰石纳米颗粒，已被开发出来并用于蛋白质分离和磷酸肽的选择性富集。通过将棒状羟基磷灰石纳米颗粒简单地混合在聚合混合物中，然后进行原位聚合，将棒状羟基磷灰石纳米颗粒掺入到聚(甲基丙烯酸2-羟乙酯-共聚-二甲基丙烯酸乙二酯)整料中。详细探讨了聚合混合物中单体和羟基磷灰石纳米颗粒的百分比对整体柱性能的影响。我们发现整体的负载能力与其他羟基磷灰石分离介质相当。然而，由于快速的质量传输，这些柱的使用速度更高。通过分离包括卵清蛋白、肌红蛋白、溶菌酶和细胞色素 c 在内的蛋白质模型混合物以及单克隆抗体及其与蛋白 A 的聚集体，证明了整体柱的功能。还使用羟基磷灰石整体柱实现了从卵清蛋白、α-酪蛋白和 β-酪蛋白消化物的复杂肽混合物中捞出的磷酸肽的选择性富集和 MALDI/MS 表征。
New monolithic capillary columns with embedded commercial hydroxyapatite nanoparticles have been developed and used for protein separation and selective enrichment of phosphopeptides. The rod-shaped hydroxyapatite nanoparticles were incorporated into the poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) monolith by simply admixing them in the polymerization mixture followed by in situ polymerization. The effect of percentages of monomers and hydroxyapatite nanoparticles in the polymerization mixture on the performance of the monolithic column was explored in detail. We found that the loading capacity of the monolith is on par with other hydroxyapatite separation media. However, the speed at which these columns can be used is higher due to the fast mass transport. The function of the monolithic columns was demonstrated with the separations of a model mixture of proteins including ovalbumin, myoglobin, lysozyme, and cytochrome c as well as a monoclonal antibody and its aggregates with protein A. Selective enrichment and MALDI/MS characterization of phosphopeptides fished-out from complex peptide mixtures of ovalbumin, alpha-casein, and beta-casein digests were also achieved using the hydroxyapatite monolith.