Institute of Organic Chemistry and Center for Molecular Biosciences (CMBI), Leopold-Franzens University Innsbruck, 6020 Innsbruck, Austria.
Institute of Biochemistry and Center for Molecular Biosciences (CMBI), Leopold-Franzens University Innsbruck, 6020 Innsbruck, Austria.
Anal Chem. 2022 Jun 21;94(24):8580-8587. doi: 10.1021/acs.analchem.1c05232. Epub 2022 Jun 9.
Additive manufacturing (3D printing) has greatly revolutionized the way researchers approach certain technical challenges. Despite its outstanding print quality and resolution, stereolithography (SLA) printing is cost-effective and relatively accessible. However, applications involving mass spectrometry (MS) are few due to residual oligomers and additives leaching from SLA-printed devices that interfere with MS analyses. We identified the crosslinking agent urethane dimethacrylate as the main contaminant derived from SLA prints. A stringent washing and post-curing protocol mitigated sample contamination and rendered SLA prints suitable for MS hyphenation. Thereafter, SLA printing was used to produce 360 μm I.D. microcolumn chips with excellent structural properties. By packing the column with polystyrene microspheres and covalently immobilizing pepsin, an exceptionally effective microscale immobilized enzyme reactor (μIMER) was created. Implemented in an online liquid chromatography-MS/MS setup, the protease microcolumn enabled reproducible protein digestion and peptide mapping with 100% sequence coverage obtained for three different recombinant proteins. Additionally, when assessing the μIMER digestion efficiency for complex proteome samples, it delivered a 144-fold faster and significantly more efficient protein digestion compared to 24 h for bulk digestion. The 3D-printed μIMER withstands remarkably high pressures above 130 bar and retains its activity for several weeks. This versatile platform will enable researchers to produce tailored polymer-based enzyme reactors for various applications in analytical chemistry and beyond.
增材制造（3D 打印）极大地改变了研究人员处理某些技术挑战的方式。尽管立体光固化（SLA）打印具有出色的打印质量和分辨率，但它具有成本效益，并且相对容易获得。然而，由于 SLA 打印设备中残留的低聚物和添加剂会浸出并干扰 MS 分析，因此涉及质谱（MS）的应用很少。我们确定了交联剂聚氨酯二甲基丙烯酸酯是源自 SLA 打印的主要污染物。严格的清洗和后固化方案减轻了样品污染，使 SLA 打印品适合 MS 联用。此后，使用 SLA 打印制作了具有出色结构性能的 360μm ID 微柱芯片。通过将聚苯乙烯微球填充到柱中并共价固定胃蛋白酶，创建了一种非常有效的微尺度固定化酶反应器（μIMER）。在在线液相色谱-MS/MS 装置中实施时，蛋白酶微柱能够实现可重复的蛋白质消化和肽图绘制，对于三种不同的重组蛋白均获得了 100%的序列覆盖率。此外，在评估 μIMER 对复杂蛋白质组样品的消化效率时，与批量消化 24 小时相比，其实现了 144 倍的快速消化和显著更高的效率。3D 打印的 μIMER 可承受超过 130 巴的高压力，并保持数周的活性。这个多功能平台将使研究人员能够为分析化学和其他领域的各种应用生产定制的聚合物基酶反应器。
