Abstract Portable microreactors (PMRs) have valuable applications in the field of proteomics. However, no PMRs have been developed for the size-selective proteolysis of low molecular-weight proteins (LWPs) with mass less than 30 kDa, which are of great importance in biomarkers, biomolecular signaling and the field of pathology. In this study, a novel syringe-filter-based PMR using core-shell mesoporous silica (CSMS) microspheres as an enzyme support was reported. Because of the unique properties of CSMS microspheres, the PMR was easily fabricated by on-site physical adsorption even without any incubation, which was accomplished by simply pushing trypsin solution through a syringe filter containing the microspheres. Efficient immobilization of trypsin was demonstrated by characterizing the PMR using various spectroscopic techniques. The PMR loading amount was approximately 2.9 mg/g trypsin/CSMS microspheres with negligible leaching. The capability of the proposed PMR for size-selective proteolysis was assessed by investigating the digestion of proteins with different molecular weights, i.e., cytochrome C (12.38 kDa), lysozyme (14.39 kDa), myoglobin (17.00 kDa) and ferritin (440.00 kDa). The results of capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) analysis showed that, by using the proposed PMR, the peptide numbers and sequence coverage of LWPs in the sample were enhanced owing to the size-exclusion interaction of the mesopores against large proteins. The proposed PMRs had essential features of easy fabrication, low cost, reusability and efficient size-selective proteolysis, and performing multiple functions including size-exclusion of LWPs, trypsin digestion and filtration in a single injection step of protein samples. This study provided a robust approach for fabricating PMRs that could perform rapid size-selective digestion of LWPs.

中文翻译：

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一种基于注射器过滤器的便携式微反应器，用于低分子量蛋白质的尺寸选择性蛋白水解

摘要 便携式微反应器（PMR）在蛋白质组学领域有着重要的应用。然而，尚未开发出用于质量小于 30 kDa 的低分子量蛋白质 (LWP) 的大小选择性蛋白水解的 PMR，这在生物标志物、生物分子信号传导和病理学领域具有重要意义。在这项研究中，报道了一种使用核壳介孔二氧化硅 (CSMS) 微球作为酶支持物的新型基于注射器过滤器的 PMR。由于 CSMS 微球的独特性质，即使没有任何孵育，也可以通过现场物理吸附轻松制造 PMR，只需将胰蛋白酶溶液推过含有微球的注射器过滤器即可完成。通过使用各种光谱技术表征 PMR，证明了胰蛋白酶的有效固定。PMR 加载量为约 2.9 mg/g 胰蛋白酶/CSMS 微球，浸出可忽略不计。通过研究不同分子量的蛋白质，即细胞色素 C (12.38 kDa)、溶菌酶 (14.39 kDa)、肌红蛋白 (17.00 kDa) 和铁蛋白 (440.00 kDa) 的消化，评估了提议的 PMR 的大小选择性蛋白水解能力. 毛细管电泳-电喷雾电离-质谱 (CE-ESI-MS) 分析的结果表明，通过使用所提出的 PMR，样品中 LWPs 的肽数量和序列覆盖率由于与介孔对抗大蛋白质。所提出的 PMR 具有易于制造、成本低、可重复使用和高效的尺寸选择性蛋白水解等基本特征，并具有多种功能，包括 LWP 的尺寸排除、在蛋白质样品的单个进样步骤中进行胰蛋白酶消化和过滤。这项研究为制造 PMR 提供了一种可靠的方法，可以对 LWP 进行快速的尺寸选择性消化。