Hahn Hans W, Rainer Matthias, Ringer Thomas, Huck Christian W, Bonn Günther K
Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innsbruck, Austria.
J Proteome Res. 2009 Sep;8(9):4225-30. doi: 10.1021/pr900188x.
Trypsin was immobilized on glycidylmethacrylate-co-divinylbenzene (GMA/DVB) polymerized in pipet tips for online enzymatic digestion of proteins. The major advantages of in-tip digestion are easy handling and small sample amount required for analysis. Microwave-assisted digestion was applied for highly efficient and time saving proteolysis. Adaption to an automated robotic system allowed fast and reproducible sample treatment. Investigations with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and liquid chromatography coupled with electrospray-ionization mass spectrometry (LC-ESI/MS) attested high sequence coverages (SCs) for the three standard proteins, myoglobin (Myo, 89%), bovine serum albumin (BSA, 78%) and alpha-casein (alpha-Cas, 83%). Compared to commercially available trypsin tips, clear predominance concerning the digestion performance was achieved. Storageability was tested over a period of several weeks and results showed only little decrease (<5%) of protein sequence coverages. The application of microwave-assisted in-tip digestion (2 min) with full automation by a robotic system allows high-throughput analysis (96 samples within 80 min) and highly effective proteolysis.
将胰蛋白酶固定在移液器吸头中聚合的甲基丙烯酸缩水甘油酯-二乙烯基苯共聚物（GMA/DVB）上，用于蛋白质的在线酶解。吸头内消化的主要优点是操作简便，分析所需样品量少。采用微波辅助消化实现高效省时的蛋白水解。适配自动化机器人系统可实现快速且可重复的样品处理。通过基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF/MS）和液相色谱-电喷雾电离质谱（LC-ESI/MS）进行的研究表明，三种标准蛋白，即肌红蛋白（Myo，89%）、牛血清白蛋白（BSA，78%）和α-酪蛋白（α-Cas，83%）的序列覆盖率很高。与市售胰蛋白酶吸头相比，在消化性能方面具有明显优势。在数周时间内测试了储存稳定性，结果表明蛋白质序列覆盖率仅略有下降（<5%）。通过机器人系统实现完全自动化的微波辅助吸头内消化（2分钟）可实现高通量分析（80分钟内分析96个样品）和高效蛋白水解。
